Metal-binding compounds and uses therefor

ABSTRACT

The invention provides a method of reducing the damage done by reactive oxygen species (ROS) in an animal. The invention also provides a method of reducing the concentration of a metal in an animal. These methods comprise administering to the animal an effective amount of a metal-binding compound as further described in the application. The invention further provides a method of reducing the damage done by ROS to a cell, a tissue or an organ that has been removed from an animal. This method comprising contacting the cell, tissue or organ with a solution or medium containing an effective amount of a metal-binding compound of the invention. The invention further provides novel metal-binding compounds, pharmaceutical compositions comprising the metal-binding compounds, and kits comprising a container holding a metal-binding compound of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending application Ser.No. 09/678,202, filed Sep. 29, 2000. This application also claimsbenefit of provisional applications 60/283,507, filed Apr. 11, 2001,60/281,648, filed Apr. 4, 2001, 60/______, (originally given applicationSer. No. 09/816,679), filed Mar. 22, 2001, 60/157,404, filed Oct. 1,1999, 60/211,078, filed Jun. 13, 2000, and 60/268,558, filed Feb. 13,2001. The entire disclosure of the aforementioned applications isconsidered to be part of the disclosure of this application and ishereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a method of reducing the molecular, cellularand tissue damage done by reactive oxygen species (ROS). The inventionalso relates to certain compounds, especially certain peptides andpeptide derivatives, that bind metal ions, particularly Cu(II). Thebinding of metal ions by the compounds of the invention inhibits theformation and/or accumulation of ROS and/or targets the damage done byROS to the compounds themselves (i.e., the compounds of the inventionmay act as sacrificial antioxidants). The compounds of the invention canalso be used to reduce the concentration of a metal in an animal in needthereof.

BACKGROUND

Reactive oxygen species (ROS) include free radicals (e.g., superoxideanion and hydroxyl, peroxyl, and alkoxyl radicals) and non-radicalspecies (e.g., singlet oxygen and hydrogen peroxide). ROS are capable ofcausing extensive cellular and tissue damage, and they have beenreported to play a major role in a variety of diseases and conditions.Indeed, ROS have been implicated in over 100 diseases and pathogenicconditions, and it has been speculated that ROS may constitute a commonpathogenic mechanism involved in all human diseases. Stohs, J. BasicClin. Physiol. Pharmacol, 6, 205-228 (1995). For reviews describing ROS,their formation, the mechanisms by which they cause cellular and tissuedamage, and their involvement in numerous diseases and disorders, see,e.g., Manso, Rev. Port. Cardiol., 11, 997-999 (1992); Florence, Aust. NZ J. Opthalmol., 23, 3-7 (1992); Stohs, J. Basic Clin. Physiol.Pharmacol., 6, 205-228 (1995); Knight, Ann. Clin. Lab. Sci., 25, 111-121(1995); Kerr et al., Heart & Lung, 25, 200-209 (1996); Roth, Acta Chir.Hung., 36, 302-305 (1997).

Ischemia/reperfusion is the leading cause of illness and disability inthe world. Cardiovascular ischemia, in which the body's capacity toprovide oxygen to the heart is diminished, is the leading cause ofillness and death in the United States. Cerebral ischemia is a precursorto cerebrovascular accident (stroke), which is the third leading causeof death in the United States. Ischemia also occurs in other organs(e.g., kidney, liver, lung, and the intestinal tract), in harvestedorgans (e.g., organs harvested for transplantation or for research(e.g., perfused organ models)), and as a result of surgery where bloodflow is interrupted (e.g., open heart surgery and coronary bypasssurgery). Ischemia need not be limited to one organ; it can also be moregeneralized (e.g., in hemorrhagic shock).

Cellular and tissue damage occur during ischemia as result of oxygendeficiency. However, the damage that occurs during ischemia is generallylight compared to the severe damage that occurs upon reperfusion ofischemic tissues and organs. See, e.g., Manso, Rev. Port. Cardiol., 11,997-999 (1992); Stohs, J. Basic Clin. Physiol. Pharmacol., 6, 205-228(1995); Knight, Ann. Clin. Lab. Sci., 25, 111-121 (1995); Kerr et al.,Heart & Lung, 25, 200-209 (1996); Roth, Acta Chir. Hung., 36, 302-305(1997). ROS have been reported to be responsible for the severe damagecaused by reperfusion of ischemic tissues and organs. See, e.g., Manso,Rev. Port. Cardiol., 11, 997-999 (1992); Stohs, J. Basic Clin. Physiol.Pharmacol., 6, 205-228 (1995); Knight, Ann. Clin. Lab. Sci., 25, 111-121(1995); Kerr et al., Heart & Lung, 25, 200-209 (1996); Roth, Acta Chir.Hung., 36, 302-305 (1997).

Metal ions, primarily transition metal ions, can cause the productionand accumulation of ROS. In particular, copper and iron ions releasedfrom storage sites are one of the main causes of the production of ROSfollowing injury, including ischemia/reperfusion injury and injury dueto heat, cold, trauma, excess exercise, toxins, radiation, andinfection. Roth, Acta Chir. Hung., 36, 302-305 (1997). Copper and ironions, as well as other transition metal ions (e.g., vanadium, andchromium ions), have been reported to catalyze the production of ROS.See, e.g., Stohs, J. Basic Clin. Physiol. Pharmacol., 6, 205-228 (1995);Halliwell et al., Free Radicals In Biology And Medicine, pages 1-19(Oxford University 1989); Marx et al., Biochem. J., 236, 397-400 (1985);Quinlan et al., J. Pharmaceutical Sci., 81, 611-614 (1992). Othertransition metal ions (e.g., cadmium, mercury, and nickel ions) andother metal ions (e.g., arsenic and lead ions) have been reported todeplete some of the molecules of the natural antioxidant defense system,thereby causing an increased accumulation of ROS. See, e.g., Stohs, J.Basic Clin. Physiol. Pharmacol., 6, 205-228 (1995). Although it has beenreported that free copper ions bind nonspecifically to the amino groupsof essentially any protein (Gutteridge et al., Biochim. Biophys. Acta,759, 38-41 (1983)), copper ions bound to proteins can still cause theproduction of ROS which damage at least the protein to which the copperions are bound. See, e.g., Gutteridge et al., Biochim. Biophys. Acta,759, 38-41 (1983); Marx et al., Biochem. J., 236, 397-400 (1985);Quinlan et al., J. Pharmaceutical Sci., 81, 611-614 (1992).

Albumin has been characterized as an extracellular antioxidant. See,e.g., Halliwell and Gutteridge, Arch. Biochem. Biophys., 280, 1-8(1990); Das et al., Methods Enzymol., 233, 601-610 (1994); Stohs, J.Basic Clin. Physiol. Pharmacol., 6, 205-228 (1995); Dunphy et al., Am.J. Physiol., 276, H1591-H1598 (1999)). The antioxidant character ofalbumin has been attributed to several of albumin's many physiologicalfunctions, including albumin's ability to bind metals (particularlycopper ions), to bind fatty acids, to bind and transport steroids, tobind and transport bilirubin, to scavenge HOCl, and others. See, e.g.,Halliwell and Gutteridge, Arch. Biochem. Biophys., 280, 1-8 (1990);Halliwell and Gutteridge, Arch. Biochem. Biophys., 246, 501-514 (1986);Stohs, J. Basic Clin. Physiol. Pharmacol., 6, 205-228 (1995); Dunphy etal., Am. J. Physiol., 276, H1591-H1598 (1999)). Albumin contains severalmetal binding sites, including one at the N-terminus. The N-terminalmetal-binding sites of several albumins, including human, rat and bovineserum albumins, exhibit high-affinity for Cu(II) and Ni(II), and theamino acids involved in the high-affinity binding of these metal ionshave been identified. See Laussac et al., Biochem., 23, 2832-2838(1984); Predki et al., Biochem. J., 287, 211-215 (1992); Masuoka et al.,J. Biol. Chem., 268, 21533-21537 (1993). It has been reported thatcopper bound to albumin at metal binding sites other than thehigh-affinity N-terminal site produce free radicals which causesextensive damage to albumin at sites dictated by the location of the“loose” metal binding sites, resulting in the characterization ofalbumin as a “sacrificial antioxidant.” See Marx et al., Biochem. J.,236, 397-400 (1985); Halliwell et al., Free Radicals In Biology AndMedicine, pages 1-19 (Oxford University 1989); Halliwell and Gutteridge,Arch. Biochem. Biophys., 280, 1-8 (1990); Quinlan et al., J.Pharmaceutical Sci., 81, 611-614 (1992).

Despite the foregoing, attempts to use albumin as a treatment forcerebral ischemia have shown mixed results. It has been reported thatalbumin is, and is not, neuroprotective in animal models of cerebralischemia. Compare Huh et al., Brain Res., 804, 105-113 (1998) andRemmers et al., Brain Res., 827, 237-242 (1999), with Little et al.,Neurosurgery, 9, 552-558 (1981) and Beaulieu et al., J. Cereb. BloodFlow. Metab., 18, 1022-1031 (1998).

Mixed results have also been obtained using albumin in cardioplegiasolutions for the preservation of excised hearts. As reported in Dunphyet al., Am. J. Physiol., 276, H1591-H1598 (1999), the addition ofalbumin to a standard cardioplegia solution for the preservation ofexcised hearts did not improve the functioning of hearts perfused withthe solution for twenty-four hours. Hearts did demonstrate improvedfunctioning when perfused with a cardioplegia solution containingalbumin and several enhancers (insulin, ATP, corticosterone, and pyruvicacid). This was a synergistic effect, since the enhancers alone, as wellas the albumin alone, did not significantly improve heart function. Anearlier report of improved heart function using cardioplegia solutionscontaining albumin was also attributed to synergism between enhancersand albumin. See the final paragraph of Dunphy et al., Am. J. Physiol.,276, H1591-H1598 (1999) and Hisatomi et al., Transplantation, 52,754-755 (1991), cited therein. In another study, hearts perfused with acardioplegia solution containing albumin increased reperfusion injury ina dose-related manner, as compared to a solution not containing albumin.Suzer et al., Pharmacol. Res., 37, 97-101 (1998). Based on their studyand the studies of others, Suzer et al. concluded that albumin had notbeen shown to be effective for cardioprotection. They further noted thatthe use of albumin in cardioplegia solutions could be unsafe due topossible allergic reactions and the risks associated with the use ofblood products.

Finally, although albumin has been characterized as an antioxidant, ithas also been reported to enhance superoxide anion production bymicroglia (Si et al., GLIA, 21, 413-418 (1997)). This result led theauthors to speculate that albumin leaking through the disrupted bloodbrain barrier in certain disorders potentiates the production ofsuperoxide anion by microglia, and that this increased production ofsuperoxide anion is responsible for the pathogenesis of neuronal damagein cerebral ischemia/reperfusion and some neurodegenerative diseases.

As noted above, the N-terminal metal-binding sites of several albuminsexhibit high-affinity for Cu(II) and Ni(II). These sites have beenstudied extensively, and a general amino terminal Cu(II)- andNi(II)-binding (ATCUN) motif has been identified. See, e.g., Harford andSarkar, Acc. Chem. Res., 30, 123-130 (1997). The ATCUN motif can bedefined as being present in a protein or peptide which has a free —NH₂at the N-terminus, a histidine residue in the third position, and twointervening peptide nitrogens. See, e.g., Harford and Sarkar, Acc. Chem.Res., 30, 123-130 (1997). Thus, the ATCUN motif is provided by thepeptide sequence Xaa Xaa His, where Xaa is any amino acid exceptproline. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30, 123-130(1997). The Cu(II) and Ni(II) are bound by four nitrogens provided bythe three amino acids of the ATCUN motif (the nitrogen of the free —NH₂,the two peptide nitrogens, and an imidazole nitrogen of histidine) in aslightly distorted square planar configuration. See, e.g., Harford andSarkar, Acc. Chem. Res., 30, 123-130 (1997). Side-chain groups of thethree amino acids of which the ATCUN motif consists can be involved inthe binding of the Cu(II) and Ni(II), and amino acids near these threeN-terminal amino acids may also have an influence on the binding ofthese metal ions. See, e.g., Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997); Bal et al., Chem. Res. Toxicol., 10, 906-914 (1997). Forinstance, the sequence of the N-terminal metal-binding site of humanserum albumin is Asp Ala His Lys [SEQ ID NO:1], and the free side-chaincarboxyl of the N-terminal Asp and the Lys residue have been reported tobe involved in the binding of Cu(II) and Ni(II), in addition to the fournitrogens provided by Asp Ala His. See Harford and Sarkar, Acc. Chem.Res., 30, 123-130 (1997); Laussac et al., Biochem., 23, 2832-2838(1984); and Sadler et al., Eur. J. Biochem., 220, 193-200 (1994).

The ATCUN motif has been found in other naturally-occurring proteinsbesides albumins, and non-naturally-occurring peptides and proteinscomprising the ATCUN motif have been synthesized. See, e.g., Harford andSarkar, Acc. Chem. Res., 30, 123-130 (1997); Bal et al., Chem. Res.Toxicol., 10, 906-914 (1997); Mlynarz, et al., Speciation 98: Abstracts,http://www.jate.u-szeged.hu/˜spec98/abstr/mlynar.html. Cu(II) and Ni(II)complexes of ATCUN-containing peptides and proteins have been reportedto exhibit superoxide dismutase (SOD) activity. See Cotelle et al., J.Inorg. Biochem., 46, 7-15 (1992); Ueda et al., J. Inorg. Biochem., 55,123-130 (1994). Despite their reported SOD activity, these complexesstill produce free radicals which damage DNA, proteins and otherbiomolecules. See Harford and Sarkar, Acc. Chem. Res., 30, 123-130(1997); Bal et al., Chem. Res. Toxicol., 10, 915-21 (1997); Ueda et al.,Free Radical Biol. Med., 18, 929-933 (1995); Ueda et al., J. Inorg.Biochem., 55, 123-130 (1994); Cotelle et al., J. Inorg. Biochem., 46,7-15 (1992). As a consequence, it has been hypothesized that at leastsome of the adverse effects of copper and nickel in vivo (e.g., causingcancer and birth defects) are attributable to the binding of Cu(II) andNi(II) to ATCUN-containing proteins which causes the production ofdamaging free radicals. See Harford and Sarkar, Acc. Chem. Res., 30,123-130 (1997); Bal et al., Chem. Res. Toxicol., 10, 915-921 (1997);Cotelle et al., J. Inorg. Biochem., 46, 7-15 (1992). Cf. Koch et al.,Chem. & Biol., 4, 549-60 (1997). The damaging effects produced by aCu(II) complex of an ATCUN-containing peptide in combination withascorbate have been exploited to kill cancer cells in vitro and toproduce anti-tumor effects in vivo. See Harford and Sarkar, Acc. Chem.Res., 30, 123-130 (1997).

SUMMARY OF THE INVENTION

The invention provides a method of reducing the damage done by reactiveoxygen species (ROS) in an animal. The method comprises administering tothe animal an effective amount of a metal-binding peptide having theformula P₁-P₂ or a physiologically-acceptable salt thereof.

The invention further provides a method of reducing the damage done byROS to a cell, a tissue or an organ that has been removed from ananimal. This method comprises contacting the cell, tissue or organ witha solution containing an effective amount of the peptide P₁-P₂ or aphysiologically-acceptable salt thereof.

The invention also provides a method of reducing the concentration of ametal in an animal in need thereof. The method comprises administeringto the animal an effective amount of a metal-binding peptide having theformula P₁-P₂ or a physiologically-acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier and the peptide P₁-P₂ or aphysiologically-acceptable salt thereof.

In addition, the invention provides a kit for reducing the damage doneby ROS to a cell, a tissue or an organ that has been removed from ananimal. The kit comprises a container holding the peptide P₁-P₂.

In the formula P₁-P₂:

-   -   P₁ is Xaa₁ Xaa₂ His or Xaa₁ Xaa₂ His Xaa₃; and    -   P₂ is (Xaa₄)_(n).

Xaa₁ is glycine (Gly), alanine (Ala), valine (Val), leucine (Leu),isoleucine (Ile), serine (Ser), threonine (Thr), aspartic acid (Asp),asparagine (Asn), glutamic acid (Glu), glutamine (Gln), lysine (Lys),hydroxylysine (Hylys), histidine (His), arginine (Arg), ornithine (Orn),phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys),methionine (Met) or α-hydroxymethylserine (HMS). In addition, Xaa₁ canbe an amino acid which comprises a δ-amino group (e.g., Orn, Lys) havinganother amino acid or a peptide attached to it (e.g., Gly (δ)-Orn). Xaa₁is preferably Asp, Glu, Arg, Thr, or HMS. More preferably, Xaa₁ is Aspor Glu. Most preferably Xaa₁ is Asp.

Xaa₂ is Gly, Ala, β-Ala, Val, Len, Ile, Ser, Thr, Asp, Asn, Glu, Gln,Lys, Hylys, His, Arg, Orn, Phe, Tyr, Trp, Cys, Met or HMS. Xaa₂ ispreferably Gly, Ala, Val, Leu, Ile, Thr, Ser, Asn, Met, His or HMS. Morepreferably Xaa₂ is Ala, Val, Thr, Ser, Leu, or HMS. Even more preferablyXaa₂ is Ala, Thr, Leu, or HMS. Most preferably Xaa₂ is Ala.

Xaa₃ is Gly, Ala, Val, Lys, Arg, Orn, Asp, Glu, Asn, Gln, or Trp,preferably Lys.

Xaa₄ is any amino acid.

Finally, n is 0-100, preferably 0-10, more preferably 0-5, and mostpreferably 0.

In a preferred embodiment, at least one of the amino acids of P₁, otherthan β-Ala when it is present, is a D-amino acid. Preferably, theD-amino acid is Xaa₁, His, or both. Most preferably all of the aminoacids of P₁, other than β-Ala when it is present, are D-amino acids. Inanother preferred embodiment, at least one of the amino acids of P₁,other than β-Ala when it is present, is a D-amino acid, and at least 50%of the amino acids of P₂ are also D-amino acids. Most preferably all ofthe amino acids of P₂ are D-amino acids.

In another preferred embodiment, at least one amino acid of P₁ and/or P₂is substituted with (a) a substituent that increases the lipophilicityof the peptide without altering the ability of P₁ to bind metal ions,(b) a substituent that protects the peptide from proteolytic enzymeswithout altering the ability of P₁ to bind metal ions, or (c) asubstituent which is a non-peptide, metal-binding functional group thatimproves the ability of the peptide to bind metal ions.

The invention provides another method of reducing the damage done by ROSin an animal. The method comprises administering to the animal aneffective amount of a metal-binding peptide (MBP) having attachedthereto a non-peptide, metal-binding functional group. The metal-bindingpeptide MBP may be any metal-binding peptide, not just P₁-P₂.

The invention further provides another method of reducing the damagedone by ROS to a cell, a tissue or an organ that has been removed froman animal. This method comprises contacting the cell, tissue or organwith a solution containing an effective amount of a metal-bindingpeptide MBP having attached thereto a non-peptide, metal-bindingfunctional group.

The invention provides another method of reducing the concentration of ametal in an animal in need thereof. The method comprises administeringto the animal an effective amount of a metal-binding peptide MBP havingattached thereto a non-peptide, metal-binding functional group.

The invention also provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a metal-binding peptide MBPhaving attached thereto a non-peptide, metal-binding functional group.

The invention also provides a kit for reducing the damage done by ROS toa cell, a tissue or an organ that has been removed from an animal. Thekit comprises a container holding a metal-binding peptide MBP havingattached thereto a non-peptide, metal-binding functional group.

The invention provides yet another method of reducing the damage done byreactive oxygen species (ROS) in an animal. The method comprisesadministering to the animal an effective amount of a metal-bindingpeptide dimer of the formula P₃-L-P₃, wherein each P₃ may be the same ordifferent and is a peptide which is capable of binding a metal ion, andL is a chemical group which connects the two P₃ peptides through theirC-terminal amino acids. In a preferred embodiment, one or both of thetwo P₃ peptides is P₁.

The invention further provides a method of reducing the damage done byROS to a cell, a tissue or an organ that has been removed from ananimal. This method comprises contacting the cell, tissue or organ witha solution containing an effective amount of the metal-binding peptidedimer of the formula P₃-L-P₃.

The invention also provides a method of reducing the concentration of ametal in an animal in need thereof. The method comprises administeringto the animal an effective amount of the metal-binding peptide dimer ofthe formula P₃-L-P₃.

The invention also provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier and the metal-binding peptide dimerof the formula P₃-L-P₃.

In addition, the invention provides a kit for reducing the damage doneby ROS to a cell, a tissue or an organ that has been removed from ananimal. The kit comprises a container holding the metal-binding peptidedimer of the formula P₃-L-P₃.

In addition, the invention provides a peptide having the formula P₁-P₂,or a physiologically-acceptable salt thereof, wherein at least one aminoacid of P₁, other than β-Ala when it is present, is a D-amino acid.

Further provided by the invention is a peptide having the formula P₁-P₂,or a physiologically-acceptable salt thereof, wherein at least one aminoacid of P₁ and/or P₂ is substituted with (a) a substituent thatincreases the lipophilicity of the peptide without altering the abilityof P₁ to bind metal ions, (b) a substituent that protects the peptidefrom proteolytic enzymes without altering the ability of P₁ to bindmetal ions, or (c) a substituent which is a non-peptide, metal-bindingfunctional group that improves the ability of the peptide to bind metalions.

In addition, the invention provides a peptide having the formula P₁-P₂,wherein P₁ is defined above, and P₂ is a peptide sequence whichcomprises the sequence of a metal-binding site.

The invention also provides a metal-binding peptide MBP having attachedthereto a non-peptide, metal-binding functional group.

Finally, the invention provides the metal-binding peptide dimer of theformula P₃-L-P₃.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D: Formulas of tetrapeptide Asp Ala His Lys [SEQ ID NO: 1]showing points of possible substitution.

FIGS. 2A-B: Schematic diagrams of the synthesis of derivatives of thetetrapeptide Asp Ala His Lys [SEQ ID NO:1] coming within the formula ofFIG. 1C (FIG. 2A) and FIG. 1B (FIG. 2B).

FIG. 3A-B: Formulas of cyclohexane diamine derivatives.

FIGS. 3C-D: Schematic diagrams of syntheses of cyclohexane diaminederivatives of the tetrapeptide Asp Ala His Lys [SEQ ID NO:1].

FIG. 4: Formula of a tetraacetic acid derivative of the tetrapeptide AspAla His Lys [SEQ ID NO:1].

FIG. 5: Formula of a bispyridylethylamine derivative of the tetrapeptideAsp Ala His Lys [SEQ ID NO:1].

FIGS. 6A-B: Formulas of mesoporphyrin IX with (FIG. 6B) and without(FIG. 6A) a bound metal ion M.

FIG. 6C: Formula of mesoporphyrin IX derivative of the tetrapeptide AspAla His Lys [SEQ ID NO:1].

FIG. 7: Formulas of monosaccharides.

FIG. 8: Diagram of a parabiotic blood perfusion system in which anisolated heart is perfused in the Langendorff mode with blood at 37° C.from a support animal of the same species.

FIG. 9: Diagram of the treatments of isolated perfused hearts with drugand saline in the parabiotic blood perfusion system illustrated in FIG.8.

FIG. 10: Graph of contracture versus duration of ischemia showing theeffect of a drug (D-Asp D-Ala D-His D-Lys) on contracture duringischemia in the blood-perfused rat heart model illustrated in FIGS. 8and 9. In FIG. 10, —□— is saline control, and —∘— is drug.

FIG. 11: Graph of left ventricle diastolic pressure (LVDP; expressed asa percentage of the 20-minute pre-intervention baseline value) versusduration of reperfusion showing the effect of the drug D-Asp D-Ala D-HisD-Lys on post-ischemic recovery of LVDP in the blood-perfused rat heartmodel illustrated in FIGS. 8 and 9. * indicates p≦0.05. In FIG. 11, —□—is saline control, and —∘— is drug.

FIG. 12: Graph of left ventricle end diastolic pressure (LVEDP) versusduration of reperfusion showing the effect of the drug D-Asp D-Ala D-HisD-Lys on post-ischemic recovery of LVEDP in the blood-perfused rat heartmodel illustrated in FIGS. 8 and 9. * indicates p≦0.05. In FIG. 12, —□—is saline control, and —∘— is drug.

FIG. 13: Graph of heart rate (expressed as a percentage of the 20-minutepre-intervention baseline value) versus duration of reperfusion showingthe effect of the drug D-Asp D-Ala D-His D-Lys on post-ischemic recoveryof heart rate in the blood-perfused rat heart model illustrated in FIGS.8 and 9. * indicates p≦0.05. In FIG. 13, —□— is saline control, and —∘—is drug.

FIG. 14: Graph of perfusion pressure (expressed as a percentage of the20-minute pre-intervention baseline value) versus duration ofreperfusion showing the effect of the drug D-Asp D-Ala D-His D-Lys onpost-ischemic recovery of perfusion pressure in the blood-perfused ratheart model illustrated in FIGS. 8 and 9. In FIG. 14, —□— is salinecontrol, and —∘— is drug.

FIG. 15A-B: Graphs of absorbance at 532 nm (A532) versus incubation timein an assay for the production of hydroxyl radicals. In FIG. 15A,▪=ascorbate only, ♦=copper and ascorbate, ▴=tetrapeptide (L-Asp L-AlaL-His L-Lys [SEQ ID NO:1]), copper and ascorbate (tetrapeptide/copperratio of 1:1), X=tetrapeptide, copper and ascorbate (tetrapeptide/copperratio of 2:1). In FIG. 15B, ♦=copper and ascorbate and ▪=tetrapeptide,copper and ascorbate (tetrapeptide/copper ratio of 2:1).

FIG. 16: Graph of % inhibition versus concentration tetrapeptide (L-AspL-Ala L-His L-Lys [SEQ ID NO:1])-copper complex at a tetrapeptide/copperratio of 1:1 in the xanthine oxidase assay for superoxide dismutaseactivity.

FIG. 17: Graph of absorbance at 560 nm (A560) versus time in an assayfor superoxide radical production. In FIG. 17, ♦=ascorbate only,♦=copper and ascorbate, Δ=tetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ IDNO:1]), copper and ascorbate (tetrapeptide/copper ratio of 1:1),X=tetrapeptide, copper and ascorbate (tetrapeptide/copper ratio of 2:1).

FIG. 18: Gel after electrophoresis of DNA treated in various ways. Lane1-17 μg/ml plasmid DNA (untreated control); Lane 2-17 μg/ml plasmid DNAand 50 μM CuCl₂; Lane 3-17 μg/ml plasmid DNA and 2.5 mM ascorbate; Lane4-17 μg/ml plasmid DNA, 2.5 mM ascorbate, 50 μM CuCl₂, and 200 μMtetrapeptide (L-Asp L-Ala L-His L-Lys [SEQ ID NO:1]) (4:1 ratiotetrapeptide/copper); Lane 5-17 μg/ml plasmid DNA, 2.5 mM ascorbate, 50μM CuCl₂, and 100 μM tetrapeptide (2:1 ratio tetrapeptide/copper); Lane6-17 μg/ml plasmid DNA, 2.5 mM ascorbate, 50 μM CuCl₂, and 50 μMtetrapeptide (1:1 ratio tetrapeptide/copper); Lane 7-17 μg/ml plasmidDNA, 2.5 mM ascorbate, 50 μM CuCl₂, and 25 μM tetrapeptide (1:2 ratiotetrapeptide/copper); Lane 8-17 μg/ml plasmid DNA, 2.5 mM ascorbate, 50μM CuCl₂, and 12.5 μM tetrapeptide (1:4 ratio tetrapeptide/copper); Lane9-17 μg/ml plasmid DNA, 2.5 mM ascorbate, and 50 μM CuCl₂ (positivecontrol); and Lane 10—DNA ladder.

FIG. 19A: Formulas of peptide dimers according to the invention.

FIGS. 19B-C: Diagrams illustrating the synthesis of peptide dimersaccording to the invention.

FIG. 20: TAE (tris acetic acid EDTA (ethylenediamine tetracetic acid))agarose gel visualized with ethidium bromide showing attenuation ofROS-induced DNA double strand breaks in genomic DNA by D-Asp Ala HisLys. Lane 1—no treatment; Lane 2—CuCl₂, 50 μM; Lane 3—ascorbic acid, 100μM; Lane 4—D-Asp Ala His Lys, 200 μM; Lane 5—CuCl₂, 10 μM+ascorbic acid,50 μM; Lane 6—CuCl₂, 25 μM+ascorbic acid, 50 μM; Lane 7—CuCl₂, 50μM+ascorbic acid, 50 μM; Lane 8—CuCl₂, 50 μM+ascorbic acid, 25 μM; Lane9—CuCl₂, 50 μM+ascorbic acid, 100 μM; Lane 10—CuCl₂, 50 μM+ascorbicacid, 100 μM+D-Asp Ala His Lys, 50 μM; Lane 11—CuCl₂, 50 μM+ascorbicacid, 100 μM+D-Asp Ala His Lys, 100 μM; Lane 12—CuCl₂, 50 μM+ascorbicacid, 100 μM+D-Asp Ala His Lys, 200 μM.

FIG. 21: TAE agarose gel visualized with ethidium bromide showingattenuation of ROS-induced DNA double strand breaks in genomic DNA byD-Asp Ala His Lys. Lane 1—no treatment; Lane 2—CuCl₂, 50 μM; Lane3—ascorbic acid, 500 μM; Lane 4-D-Asp Ala His Lys, 200 μM; Lane 5—CuCl₂,10 μM+ascorbic acid, 500 μM; Lane 6—CuCl₂, 25 μM+ascorbic acid, 500 μM;Lane 7—CuCl₂, 50 μM+ascorbic acid, 500 μM; Lane 8—CuCl₂, 50 μM+ascorbicacid, 100 μM; Lane 9—CuCl₂, 50 μM+ascorbic acid, 250 μM; Lane 10—CuCl₂,50 μM+ascorbic acid, 500 μM+D-Asp Ala His Lys, 50 μM; Lane 11—CuCl₂, 50μM+ascorbic acid, 500 μM+D-Asp Ala His Lys, 100 μM; Lane 12—CuCl₂, 50μM+ascorbic acid, 500 μM+D-Asp Ala His Lys, 200 μM.

FIG. 22: Southern Blot showing attenuation of ROS-induced DNA doublestrand breaks in telomere DNA by D-Asp Ala His Lys. Lane 1—no treatment;Lane 2—CuCl₂, 50 μM; Lane 3—ascorbic acid, 100 μM, Lane 4—D-Asp Ala HisLys, 200 μM; Lane 5—CuCl₂, 50 μM+ascorbic acid, 100 μM; Lane 6—CuCl₂, 50μM+ascorbic acid, 100 μM+D-Asp Ala His Lys, 200 μM.

FIG. 23: Southern Blot showing attenuation of ROS-induced DNA doublestrand breaks in telomere DNA by D-Asp Ala His Lys. Lane 1—no treatment;Lane 2—CuCl₂, 50 μM; Lane 3—ascorbic acid, 500 μM; Lane 4—D-Asp Ala HisLys, 200 μM; Lane 5—CuCl₂, 50 μM+ascorbic acid, 100 μM; Lane 6—CuCl₂, 50μM+ascorbic acid, 250 μM; Lane 7—CuCl₂, 50 μM+ascorbic acid, 500 μM;Lane 8—CuCl₂, 50 μM+ascorbic acid, 500 μM+D-Asp Ala His Lys, 50 μM; Lane9—CuCl₂, 50 μM+ascorbic acid, 500 μM+D-Asp Ala His Lys, 100 μM.

FIGS. 24A-C: Graphs of interleukin-8 (IL-8) concentration versus varioustreatments of Jurkat cells (all treatments, except nil and copper-onlytreatments, contained ascorbic acid in addition to the other additiveslisted on the graphs).

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS

The invention provides a peptide of the formula P₁-P₂. P₁ is Xaa₁Xaa₂His or is Xaa₁ Xaa₂ His Xaa₃, wherein Xaa₁, Xaa₂, and Xaa₃ aredefined above. P₁ is a metal-binding peptide sequence that bindstransition metal ions of Groups 1b-7b or 8 of the Periodic Table ofelements (including V, Co, Cr, Mo, Mn, Ba, Zn, Hg, Cd, Au, Ag, Co, Fe,Ni, and Cu) and other metal ions (including As, Sb and Pb). The bindingof metal ions by P₁ inhibits (i.e., reduces or prevents) the productionof ROS and/or the accumulation of ROS by these metal ions and/or targetsthe damage done by ROS that may still be produced by the bound metalions to the peptide itself. As a result, the damage that can be causedby ROS in the absence of the binding of the metal ions to P₁ is reduced.In particular, P₁ binds Cu(II), Ni(II), Co(II), and Mn(II) with highaffinity. It should, therefore, be particularly effective in reducingthe damage caused by the production and accumulation of ROS by copperand nickel.

In P₁, Xaa₁ is most preferably Asp, Xaa₂ is most preferably Ala, andXaa₃ is most preferably Lys (see above). Thus, the preferred sequencesof P₁ are Asp Ala His and Asp Ala His Lys [SEQ ID NO:1]. Most preferablythe sequence of P₁ is Asp Ala His Lys [SEQ ID NO:1]. Asp Ala His is theminimum sequence of the N-terminal metal-binding site of human serumalbumin necessary for the high-affinity binding of Cu(II) and Ni(II),and Lys has been reported to contribute to the binding of these metalions to this site. Also, Asp Ala His Lys [SEQ ID NO:1] has been found bymass spectometry to bind Fe(II) and to pass through a model of the bloodbrain barrier. Other preferred sequences for P₁ include Thr Leu His (theN-terminal sequence of human α-fetoprotein), Arg Thr His (the N-terminalsequence of human sperm protamin HP2) and HMS HMS His (a syntheticpeptide reported to form extremely stable complexes with copper; seeMlynarz et al., Speciation 98: Abstracts,http://vvww.jate.u-szeged.hu/spec98/abstr/mlynar.html, Apr. 21, 1998).

P₂ is (Xaa₄)_(n), wherein Xaa₄ is any amino acid and n is 0-100. When nis large (n>about 20), the peptides will reduce the damage done by ROSextracellularly. Smaller peptides are better able to enter cells, andsmaller peptides can, therefore, be used to reduce the damage done byROS both intracellularly and extracellularly. Smaller peptides are alsoless subject to proteolysis. Therefore, in P₂, preferably n is 0-10,more preferably n is 0-5, and most preferably n is 0. Although P₂ mayhave any sequence, P₂ preferably comprises a sequence which (1) binds atransition metal, (2) enhances the ability of the peptide to penetratecell membranes and/or reach target tissues (e.g., to be able to crossthe blood brain barrier), or (3) otherwise stabilizes or enhances theperformance of the peptide. P₂ together with P₁ may also be theN-terminal sequence of a protein having an N-terminal metal-binding sitewith high affinity for copper and nickel, such as human, rat or bovineserum albumin. In the case where n=100, the peptide would have thesequence of approximately domain 1 of these albumins.

The sequences of many peptides which comprise a binding site fortransition metal ions are known. See, e.g., U.S. Pat. Nos. 4,022,888,4,461,724, 4,665,054, 4,760,051, 4,767,753, 4,810,693, 4,877,770,5,023,237, 5,059,588, 5,102,990, 5,118,665, 5,120,831, 5,135,913,5,145,838, 5,164,367, 5,591,711, 5,177,061, 5,214,032, 5,252,559,5,348,943, 5,443,816, 5,538,945, 5,550,183, 5,591,711, 5,690,905,5,759,515, 5,861,139, 5,891,418, 5,928,955, and 6,017,888, PCTapplications WO 94/26295, WO 99/57262 and WO 99/67284, European Patentapplication 327263, Lappin et al., Inorg. Chem., 17, 1630-34 (1978),Bossu et al., Inorg. Chem., 17, 1634-40 (1978), Chakrabarti, ProteinEng., 4, 57-63 (1990), Adman, Advances In Protein Chemistry, 42, 145-97(1991), Cotelle et al., J. Inorg. Biochem., 46, 7-15 (1992), Canters etal., FEBS, 325, 39-48 (1993), Regan, Annu. Rev. Biophys. Biomol.Struct., 22, 257-281 (1993), Ueda et al., J. Inorg. Biochem., 55, 123-30(1994), Ueda et al., Free Radical Biol. Med., 18, 929-33 (1995), Regan,TIBS, 20, 280-85 (1995), Ueda et al., Chem. Pharm. Bull., 43, 359-61(1995), Bal et al., Chem. Res. Toxicol., 10, 906-914 (1997), Bal et al.,Chem. Res. Toxicol., 10, 915-21 (1997), Koch et al., Chem. Biol., 4,549-60 (1997), Kowalik-Jankowska et al., J. Inorg. Biochem., 66, 193-96(1997), Harford and Sarkar, Acc. Chem. Res., 30, 123-130 (1997), Princeet al., TIBS, 23, 197-98 (1998), Mlynarz, et al., Speciation 98:Abstracts, http://www.jate.u-szeged.hu/˜spec98/abstr/mlynar.html, andAitken, Molec. Biotechnol., 12, 241-53 (1999), Whittal et al., ProteinScience, 9, 332-343 (2000). P₂ may comprise the sequence of one or moreof the metal-binding sites of these peptides.

When P₂ comprises a metal-binding site, it preferably has a sequencewhich includes a short spacer sequence between P₁ and the metal bindingsite of P₂, so that the metal-binding sites of P₁ and P₂ may potentiallycooperatively bind metal ions (similar to a 2:1 peptide:metal complex;see Example 10). Preferably, the spacer sequence is composed of 1-5,preferably 1-3, neutral amino acids. Thus, the spacer sequence may beGly, Gly Gly, Gly Ala Gly, Pro, Gly Pro Gly, etc.

In particular, when P₂ comprises a metal-binding site, it preferablycomprises one of the following sequences: (Xaa₄)_(m) Xaa₅ Xaa₂ His Xaa₃or (Xaa₄)_(m) Xaa₅ Xaa₂ His. Xaa₂, Xaa₃ and Xaa₄ are defined above, andm is 0-5, preferably 1-3. The Xaa₄ amino acid(s), if present, form(s) ashort spacer sequence between P₁ and the metal binding site of P₂ sothat the metal-binding sites of P₁ and P₂ may cooperatively bind metalions, and Xaa₄ is preferably a neutral amino acid (see the previousparagraph). Xaa₅ is an amino acid which comprises a δ-amino group(preferably Orn or Lys, more preferably Orn) having the Xaa₄ aminoacid(s), if present, or P₁ attached to it by means of the δ-amino group.See Harford and Sarkar, Acc. Chem. Res., 30, 123-130 (1997) andShullenberger et al., J. Am. Chem. Soc., 115, 11038-11039 (1993) (as aresult of this means of attachment, the α-amino group of Xaa₅ can stillparticipate in binding metals by means of the ATCUN motif). Thus, forinstance, P₁-P₂ could be Asp Ala His Gly Gly (δ)-Orn Ala His [SEQ IDNO:2].

In addition, P₂ may comprise one of the following sequences: [(Xaa₄)_(m)Xaa₅ Xaa₂ His Xaa₃]_(r), [(Xaa₄)_(m) Xaa₅ Xaa₂ His]_(r), [(Xaa₄)_(m)Xaa₅ Xaa₂ His Xaa₃ (Xaa₄)_(m) Xaa₅ Xaa₂ His]_(r), and [(Xaa₄)_(m) Xaa₅Xaa₂His(Xaa₄)_(m) Xaa₅ Xaa₂ His Xaa₃]_(r), wherein Xaa₂, Xaa₃, Xaa₄,Xaa₅ and m are defined and described above, and r is 2-100. In thismanner metal-binding polymers may be formed.

In another preferred embodiment, P₂ comprises a peptide sequence thatcan bind Cu(I). As discussed in more detail below, Cu(II) is convertedto Cu(I) in the presence of ascorbic acid or other reducing agents, andthe Cu(I) reacts with oxygen to produce ROS (see equations in Examples10 and 11). P₁ can bind Cu(II) tightly (see above) and is very effectiveby itself in inhibiting the production of ROS by copper (see Examples7-11). However, as can be seen from the equations in Examples 10 and 11,it would be desirable to also employ a P₂ which could bind Cu(I).

Peptide sequences which can bind Cu(I) are known in the art. See, e.g,Pickering et al., J. Am. Chem. Soc., 115, 9498-9505 (1993); Winge etal., in Bioinorganic Chemistry Of Copper, pages 110-123 (Karlin andTyeklar, eds., Chapman & Hall, New York, N.Y., 1993); Koch et al., Chem& Biol., 4, 549-560 (1997); Cobine et al., in Copper Transport And ItsDisorders, pages 153-164 (Leone and Mercer eds., Kluwer Academic/PlenumPublishers, New York, N.Y., 1999). These sequences include:

Met Xaa₄ Met, Met Xaa₄ Xaa₄ Met, Cys Cys, Cys Xaa₄ Cys, Cys Xaa₄ Xaa₄Cys, Met Xaa₄ Cys Xaa₄ Xaa₄ Cys, Gly Met Xaa₄ Cys Xaa₄ Xaa₄ Cys, [SEQ IDNO: 7] Gly Met Thr Cys Xaa₄ Xaa₄ Cys, [SEQ ID NO: 8] and Gly Met Thr CysAla Asn Cys, [SEQ ID NO: 9]wherein Xaa₄ is defined above. Glutathione (γ-Glu Cys Gly) is also knownto bind Cu(I). Additional Cu(I)-binding peptide sequences can beidentified using a metallopeptide combinatorial library as described in,e.g., PCT application WO 00/36136. Preferably, the Cu(I)-binding peptidecomprises the sequence Cys Xaa₄ Xaa₄ Cys (e.g., Gly Met Xaa₄ Cys Xaa₄Xaa₄ Cys [SEQ ID NO:7], more preferably Gly Met Thr Cys Xaa₄ Xaa₄ Cys[SEQ ID NO:8], most preferably Gly Met Thr Cys Ala Asn Cys [SEQ IDNO:9]).

To enhance the ability of the P₁-P₂ peptide to penetrate cell membranesand/or reach target tissues, P₂ is preferably hydrophobic or an arginineoligomer (see Rouhi, Chem. & Eng. News, 49-50 (Jan. 15, 2001)). When P₂is hydrophobic, it preferably contains 1-3 hydrophobic amino acids(e.g., Gly Gly), preferably D-amino acids. A hydrophobic P₂ may beparticularly desirable for uses of P₁-P₂ where P₁-P₂ must cross theblood brain barrier. The arginine oligomer preferably contains 6-9 Argresidues, most preferably 6-9 D-Arg residues (see Rouhi, Chem. & Eng.News, 49-50 (Jan. 15, 2001). The use of a P₂ which is an arginineoligomer may be particularly desirable when P₁-P₂ is to be administeredtopically or transdermally.

The amino acids of the peptide may be L-amino acids, D-amino acids, or acombination thereof. Preferably, at least one of the amino acids of P₁is a D-amino acid (preferably Xaa₁ and/or His), except for β-Ala, whenpresent. Most preferably, all of the amino acids of P₁, other thanβ-Ala, when present, are D-amino acids. Also, preferably about 50% ofthe amino acids of P₂ are D-amino acids, and most preferably all of theamino acids of P₂ are D-amino acids. D-amino acids are preferred becausepeptides containing D-amino acids are resistant to proteolytic enzymes,such as those that would be encountered upon administration of thepeptide to an animal (including humans) or would be present in anexcised organ perfused with a solution containing the peptide. Also, theuse of D-amino acids would not alter the ability of the peptide to bindmetal ions, including the ability of the peptide to bind copper withhigh affinity.

The peptides of the invention may be made by methods well known in theart. For instance, the peptides, whether containing L-amino acids,D-amino acids, or a combination of L- and D-amino acids, may besynthesized by standard solid-phase peptide synthesis methods. Suitabletechniques are well known in the art, and include those described inMerrifield, in Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotiseds. 1973); Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Davis etal., Biochem. Int'l, 10, 394-414 (1985); Stewart and Young, Solid PhasePeptide Synthesis (1969); U.S. Pat. Nos. 3,941,763 and 5,786,335; Finnet al., in The Proteins, 3rd ed., vol. 2, pp. 105-253 (1976); andErickson et al. in The Proteins, 3rd ed., vol. 2, pp. 257-527 (1976).See also, Polish Patent 315474 (synthesis of HMS-containing peptides)and Shullenberger et al., J. Am. Chem. Soc., 115, 1103811039 (1993)(synthesis of (δ)-Orn-containing peptides). Alternatively, the peptidesmay be synthesized by recombinant DNA techniques if they contain onlyL-amino acids. Recombinant DNA methods and suitable host cells, vectorsand other reagents for use therein, are well known in the art. See,e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1982), Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. (1989).

The invention further comprises derivatives of the peptide P₁-P₂,whether composed of L-amino acids, D-amino acids, or a combination of L-and D-amino acids, which are more resistant to proteolytic enzymes, morelipid soluble (to allow the peptides to more readily penetrate cellmembranes and/or reach target organs, such as the brain), or both. Asillustrated in FIG. 1A, P₁ can be modified in the regions indicated bythe arrows without altering the metal binding function of P₁. Inparticular, P₁ can be substituted at carbons 1 or 2 with R₁, and theterminal —COOH of P₁ can be substituted with protecting group R₂ (FIGS.1B-D). P₂ can be modified in ways similar to those described for P₁ tomake P₂ more resistant to proteolytic enzymes, more lipid soluble, orboth.

R₁ can be a straight-chain or branched-chain alkyl containing from 1 to16 carbon atoms, and the term “alkyl” includes the R and S isomers. R₁can also be an aryl or heteroaryl containing 1 or 2 rings. The term“aryl” means a compound containing at least one aromatic ring (e.g.,phenyl, naphthyl, and diphenyl). The term “heteroaryl” means an arylwherein at least one of the rings contains one or more atoms of S, N orO. These substitutions do not substantially decrease the ability of P₁to bind metal ions. In particular, the ability of P₁ to bind copper withhigh affinity is not decreased by these substitutions. For instance,some of the substituents, such as a n-butyl attached to carbon 2 (seeFIG. 1C, R₁ is n-butyl) should increase the affinity of the peptide formetal ions, such as copper, due to the inductive effect of the alkylgroup. Substitution of carbon 2 (FIG. 1C) with an aryl, heteroaryl, or along chain alkyl (about 6-16 carbon atoms) should enhance transport ofthe peptide across lipid membranes.

As noted above, methods of synthesizing peptides by solid phasesynthesis are well known. These methods can be modified to prepare thederivatives shown in FIGS. 1B-C. For example, the derivative of P₁illustrated in FIG. 1C, wherein R₁ is octyl, can be prepared asillustrated in FIG. 2A. In FIG. 2A, the elliptical element representsthe polymer resin and R_(p) is a standard carboxyl protecting group. Asillustrated in FIG. 2A, octanoic acid (freshly distilled) is treatedwith dry bromine followed by phosphorus trichloride. The mixture isheated to about 100° C. and kept at that temperature for 4 hours.α-Bromooctanoic acid is obtained as a colorless liquid upondistillation. Amination of the bromoacid is achieved by allowing theacid and an ammonia solution to stand at 40-50° C. for 30 hours. Theoctyl derivative of the amino acid is obtained by removing ammoniumbromide with methanol washes. Classical resolution methods give thedesired optically-pure D-form. Other derivatives wherein R₁ is an alkyl,aryl or heteroaryl can be prepared in the manner illustrated in FIG. 2A.

In addition, the derivative of P₁ illustrated in FIG. 1B, wherein R₁ isphenyl, can be prepared as illustrated in FIG. 2B. In FIG. 2B, Polymeris the resin, t-Bu is t-butyl, and Bz is benzyl. Other derivativeswherein R₁ is an alkyl, aryl or heteroaryl can be prepared in the mannerillustrated in FIG. 2B.

R₂ can be —NH₂, —NHR₁, —N(R₁)₂, —OR₁, or R₁ (see FIG. 1D), wherein R₁ isdefined above. These derivatives can be prepared as the last step of asolid-phase peptide synthesis before the peptide is removed from theresin by methods well known in the art. Substitutions with R₂ do notsubstantially decrease the ability of P₁ to bind metal ions.

In addition, P₁ and P₂ can be substituted with non-peptide functionalgroups that bind metal ions. These metal-binding functional groups canbe attached to one or more pendent groups of the peptide, and theresulting peptide derivatives will possess one or more sites that arecapable of binding metal ions, in addition to the binding site providedby P₁ and, optionally, the binding site provided by P₂. As aconsequence, the ability of such peptide derivatives to bind metal ionsis improved as compared to the corresponding unmodified peptide. Forinstance, the peptide derivative can bind two of the same type of metalion instead of one (e.g., two Cu(II)), the peptide derivative can bindtwo different metal ions instead of one type of metal ion (e.g., oneCu(II) and one Fe(III)), or the peptide derivative can bind one metalion better (e.g., with greater affinity) than the correspondingunmodified peptide.

Metal-binding functional groups include polyamines (e.g., diamines,triamines, etc.). Suitable diamines include 1,2-alkyldiamines,preferably alkyl diamines wherein the alkyl contains 2-10 carbon atoms(e.g., H₂N—(CH₂)—NH₂, wherein n=2-10). Suitable diamines also include1,2-aryldiamines, preferably benzene diamines (e.g.,1,2-diaminobenzene). Suitable diamines further include 1,2-cyclic alkanediamines. “Cyclic alkanes” are compounds containing 1-3 rings, eachcontaining 5-7 carbon atoms. Preferably the cyclic alkane diamine is1,2-diaminocylcohexane (cyclohexane diamine).

A particularly preferred diamine is 1,2-diaminocyclohexane (FIGS. 3A-B).Previous studies carried out by Rao & P. Williams (J. Chromatography A,693, 633 (1995)) have shown that a cyclohexane diamine derivative (FIG.3A, where PYR is pyridine) binds to a variety of metal ions. Theresulting metal chelator has been successfully used to resolve aminoacids and peptides, showing that the molecule has a very high affinityfor α-amino acids, forming a very stable coordination complex, which isunique in many respects. 1,2-Diaminocyclohexane possesses a reactiveamino functional group to which a peptide of the invention can beattached. See FIG. 3B, where M is a metal ion and at least one R₄ is-alkyl-CO-peptide, -aryl-CO-peptide, -aryl-alkyl-CO-peptide, or-alkyl-aryl-CO-peptide (see also FIGS. 3C-D). The other R₄ may be thesame or may be -alkyl-COOH, -aryl-COOH, -aryl-alkyl-COOH, oralkyl-aryl-COOH. Derivatives of the type shown in FIG. 3B will haveseveral metal-binding sites and can, therefore, be expected to bindmetal ions more readily than the unsubstituted peptide. Further, due tothe presence of the cyclohexane functionality, the compound will possesslipid-like characteristic which will aid its transport across lipidmembranes.

Cyclohexane diamine derivatives of the peptides of the invention can beprepared by two distinct routes. The first involves initial condensationwith an aldehyde followed by reduction (see FIG. 3C; in FIG. 3C Bz isbenzyl). A number of aldehydes (alkyl and aryl) react readily withcyclohexane diamine at room temperature, forming an oxime. The oxime canbe reduced with sodium borohydride under anaerobic conditions to givethe diacid derivative. The carboxyl moieties are then reacted with thefree amino groups present in carboxy-protected P₁ to give thecyclohexane diamine derivative of the peptide. The second route is adirect alkylation process which is illustrated in FIG. 3D. For example,cyclohexane diamine is treated with bromoacetic acid to give thediacetic acid derivative. The carboxyl moieties are then reacted withthe free amino groups present in carboxy-protected P₁ to give thederivative. In FIG. 3D, R₅ is H or another peptide. When R₅ is H, thederivative can be further reacted to produce typical carboxylic acidderivatives, such as esters, by methods well known in the art. Metalbinding experiments have indicated that the presence or absence of thisgroup does not have a bearing on the metal binding capacity of the wholemolecule. However, these groups would either make the moleculehydrophobic or hydrophilic, depending upon the substituent, and thismay, in turn, have an effect on delivery of the molecule acrossmembranes or to target tissues. These two synthetic routes will work forthe synthesis of diamine peptide derivatives using the other diaminesdescribed above.

Additional suitable polyamines and polyamine derivatives and methods ofattaching them to peptides are described in U.S. Pat. Nos. 5,101,041 and5,650,134, the complete disclosures of which are incorporated herein byreference. Other polyamine chelators suitable for attachment to peptidesare known. See, e.g., U.S. Pat. Nos. 5,422,096, 5,527,522, 5,628,982,5,874,573, and 5,906,996 and PCT applications WO 97/44313, WO 97/49409,and WO 99/39706.

It is well known that vicinal diacids bind to metal ions, and theaffinity for copper is particularly high. It is therefore envisaged thata peptide having a vicinal diacid functional group will be extremelyeffective in metal binding. Suitable vicinal diacids include any1,2-alkyldiacid, such as diacetic acid (succinic acid), and any1,2-aryldiacid.

The amino groups of the peptide can be reacted with diacetic acid toproduce a diacid derivative (see FIG. 4). This can be convenientlyaccomplished by reacting the amino groups of the resin-bound peptidewith a halogenated acetic acid (e.g., bromoacetic acid or chloroaceticacid) or a halogenated acetic acid derivative (e.g., benzyloxy ester).Solid phase synthetic procedures enable removal of unreacted materialsby washing with solvent. The final product is released from the resin byhydrolytic cleavage. Other diacid derivatives of the peptides of theinvention can be made in the same manner.

Polyaminopolycarboxylic acids are known to bind metals, such as copperand iron. Suitable polyaminopolycarboxylic acids for making derivativesof the peptides of the invention and methods of attaching them topeptides are described in U.S. Pat. Nos. 5,807,535 and 5,650,134, andPCT application WO 93/23425, the complete disclosures of which areincorporated herein by reference. See also, U.S. Pat. No. 5,739,395.

Vicinal polyhydroxyl derivatives are also included in the invention.Suitable vicinal polyhydroxyls include monosaccharides andpolysaccharides (i.e., disaccharide, trisaccharide, etc.). Presentlypreferred are monosaccharides. See FIG. 7. The monosaccharides fall intotwo major categories—furanoses and pyranoses. One of the prime examplesof a furanose ring system is glucose. The hydroxyl groups of glucose canbe protected as benzyl or labile t-butyloxy functional groups, whileleaving the aldehyde free to react with an amine group (e.g., that oflysine) of the tetrapeptide. Mild reduction/hydrolysis produces themonosaccharide peptide derivative. Other monosaccharide peptidederivatives can be prepared in this manner.

Bispyridylethylamine derivatives are known to form strong complexes withdivalent metal ions. When attached to the peptide, this functional groupwould provide additional chelating sites for metal ions, includingcopper. The bispyridylethyl derivative of the tetrapeptide Asp Ala HisLys [SEQ ID NO:1] is shown in FIG. 5. It is anticipated that themetal-binding capacity of this tetrapeptide derivative will be increasedby at least three-fold as compared to the underivatized peptide. Thepreparation of this bispyridylethylamine derivative shares somesimilarities with the synthesis of diacid derivatives. The two aminogroups of the tetrapeptide (one at Asp and the other at Lys) are reactedwith 2-bromoethylpyridine to give the tetra-substituted peptidederivative. The reaction is accomplished by reacting the resin-boundtetrapeptide with the bromoethylpyridine, followed by cleavage of theproduct from the resin.

Phenanthroline is another heterocyclic compound capable of bindingdivalent metal ions. Phenanthroline derivatives of the peptides can besynthesized in the same manner as for the bispyridylethylaminederivatives.

Porphyrins are a group of compounds found in all living matter andcontain a tetrapyrrolic macrocycle capable of binding to metals. Heme,chlorophyll and corrins are prime examples of this class of compoundscontaining iron, magnesium and cobalt, respectively. Mesoporphyrin IX(FIG. 6A-B, where M is a metal ion) is derived from heme and has beenobserved to possess specific affinity for copper. Addition of thisstructure to a peptide of the invention would produce aporphyrin-peptide derivative possessing several sites for binding ofcopper (see FIG. 6C). In addition to their roles in metal binding, theimidazole residues at positions 3 and 3′ of the tetrapeptide shown inFIG. 6C may provide a binding site for metals other than copper, therebystabilizing the porphyrin-metal complex. In particular, cyanocobalamine(vitamin B-12) contains cobalt as the metal in the porphyrin nucleus,and the complex is stabilized by the imidazole groups. On the basis ofthis analogy it is anticipated that the porphyrin-tetrapeptidederivative would bind cobalt (or other metals) at normal physiologicalconditions in the prophyrin nucleus and that the complex would bestabilized by the His imidazole groups.

To prepare the porphyrin-peptide derivative shown in FIG. 6C, thecarboxyl groups of mesoporphyrin IX can be activated and coupled withthe amino groups of the peptide employing standard solid-phase peptidesynthesis. Typically, the free amino group of the lysine residue of theresin-bound peptide can be coupled with carboxy activated porphyrinnucleus. The condensation product can be cleaved off the resin usingstandard methods. This method can be used to synthesize other porphyrinderivatives of peptides of the invention.

Other suitable porphyrins and macrocyclic chelators and methods ofattaching them to peptides are described in U.S. Pat. Nos. 5,994,339 and5,087,696, the complete disclosures of which are incorporated herein byreference. Other porphyrins and macrocyclic chelators that could beattached to peptides are known. See, e.g., U.S. Pat. Nos. 5,422,096,5,527,522, 5,628,982, 5,637,311, 5,874,573, and 6,004,953, PCTapplications WO 97/44313 and WO 99/39706.

A variety of additional metal chelators and methods of attaching them toproteins are described in U.S. Pat. No. 5,683,907, the completedisclosure of which is incorporated herein by reference.

Dithiocarbamates are known to bind metals, including iron. Suitabledithiocarbamates for making derivatives of the peptides of the inventionare described in U.S. Pat. Nos. 5,380,747 and 5,922,761, the completedisclosures of which are incorporated herein by reference.

Hydroxypyridones are also known to be iron chelators. Suitablehydroxypyridones for making derivatives of the peptides of the inventionare described in U.S. Pat. Nos. 4,912,118 and 5,104,865 and PCTapplication WO 98/54138, the complete disclosures of which areincorporated herein by reference.

Additional non-peptide metal chelators are known in the art or will bedeveloped. Methods of attaching chemical compounds to proteins andpeptides are well known in the art, and attaching non-peptide metalchelators to the peptides of the invention is within the skill in theart. See, e.g., those patents cited above describing such attachmentmethods.

As can be appreciated, the non-peptide, metal-binding functional groupscould be attached to another metal-binding peptide (MBP) in the samemanner as they are to peptide P₁-P₂. The resulting peptide derivativeswould contain one or more metal-binding functional groups in addition tothe metal-binding site of MBP. Preferably, MBP contains from 2-10, morepreferably 3-5, amino acids. Preferably MBP contains one or more D-aminoacids; most preferably all of the amino acids of MBP are D-amino acids.As described above, the sequences of many metal-binding peptides areknown. These peptides and peptides comprising the metal-binding sites ofthese peptides can be prepared in the same ways as described above forpeptide P₁-P₂. Derivatives of these peptides having one or moremetal-binding functional group attached to the peptide can be preparedin the same ways as described above for derivatives of peptide P₁-P₂.

The invention also provides metal-binding peptide dimers of the formula:

P₃-L-P₃.

P₃ is any peptide capable of binding a metal ion, and each P₃ may be thesame or different. Each P₃ preferably contains 2-10, more preferably3-5, amino acids. As described above, metal-binding peptides are known,and each P₃ may comprise the sequence of one or more of themetal-binding sites of these peptides. Although each P₃ may besubstituted as described above for P₁ and P₂, including with anon-peptide, metal-binding functional group, both P₃ peptides arepreferably unsubstituted. P₃ may also comprise any amino acid sequencesubstituted with a non-peptide, metal-binding functional group asdescribed above to provide the metal-binding capability of P₃.Preferably, each P₃ is an unsubstituted metal-binding peptide (i.e., anunsubstituted peptide comprising a peptide sequence which binds metalions). Most preferably, one or both of the P₃ groups is P₁ (i.e., thedimers have the sequence P₃-L-P₁, P₁-L-P₃ or, most preferably, P₁-L-P₁).P₁ is defined above.

L is a linker which is attached to the C-terminal amino acid of each P₃.L may be any physiologically-acceptable chemical group which can connectthe two P₃ peptides through their C-terminal amino acids. By“physiologically-acceptable” is meant that a peptide dimer containingthe linker L is not toxic to an animal (including a human) or an organto which the peptide dimer is administered as a result of the inclusionof the linker L in the peptide dimer. Preferably, L links the two P₃groups so that they can cooperatively bind metal ions (similar to a 2:1peptide:metal complex; see Example 10). L is also preferably neutral.Most preferably, L is a straight-chain or branched-chain alkane oralkene residue containing from 1-18, preferably from 2-8, carbon atoms(e.g., —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂(CH₃)CH₂—, —CHCH—, etc.) ora cyclic alkane or alkene residue containing from 3-8, preferably from5-6, carbon atoms (see FIG. 19A, compound D₁), preferably attached to aP₃ by means of an amide linkage. Such linkers are particularly preferredbecause they impart hydrophobicity to the peptide dimers. In anotherpreferred embodiment, L is a nitrogen-containing heterocyclic alkaneresidue (see FIG. 19A, compounds D₂, D₃ and D₄), preferably a piperazide(see FIG. 19A, compound D₂). In another preferred embodiment L is aglyceryl ester (see FIG. 19A, compound D₅; in formula D₅, R is an alkylor aryl containing, preferably containing 1-6 carbon atoms). Finally, Lcould be a metal-binding porphyrin (see FIG. 6C). These preferredlinkers L will allow the two peptides P₃ to bind metal ionscooperatively and are biocompatible, and the peptide dimers containingthese preferred linkers can be made easily and in large quantities. By“biocompatible” is meant that a peptide dimer containing the linker Ldoes not produce any undesirable side-effects due to the linker L in ananimal (including a human) or an organ to which the peptide dimer isadministered.

Methods of synthesizing the peptide dimers are illustrated in FIGS.19B-D. In general, the C-terminal amino acids (protected by methods andprotecting groups well known in the art) of the two P₃ groups areattached to L, and the resulting amino acid dimers used in standardpeptide synthetic methods to make the peptide dimers.

For instance, a peptide dimer, where each peptide has the sequence AspAla His Lys, [SEQ ID NO:1] can be synthesized by coupling protectedlysines to a free diamine functional group, either as an acid chlorideor by using standard coupling agents used in peptide synthesis (seeFIGS. 19B-C). Many suitable diamines are available commercially orsuitable diamines can be readily synthesized by methods known in theart.

For instance, the lysine dimer 2 (FIG. 19B) can be prepared as follows.To a stirred solution of 9-fluorenylmethyloxycarbonyl (Fmoc)- andt-benzyloxycarbonyl(Boc)-protected D-Lys (Fmoc-D-Lys(Boc)-OH) (20 mmole)in dry dimethylformamide (DMF; 100 mL; dry argon flushed) are addedbutane-1,4-diamine 1 and2-(1H-benzotriazole-1-yl)-1,2,3,3-tetramethyluroniumtetrafluoroborate(TBTU; 0.5 mmole). The solution is stirred for 36 hours at roomtemperature. The bis-protected lysine 2 is isolated by flashchromatography over silica and elution with mixtures of ethylacetate/methanol. The peptide dimer 3 is then prepared from theprotected lysine dimer 2 employing classical peptide synthesismethodology (see FIG. 19B).

Another peptide dimer, where each peptide has the sequence Asp Ala HisLys [SEQ ID NO:1], can be synthesized as follows. First, a differentprotected lysine dimer 4 is synthesized by acylating the two aminocenters of a piperazine 5 (see FIG. 19C; see also Chambrier et al.,Proc. Natl. Acad. Sci., 96, 10824-10829 (1999)). Then, the remainder ofthe amino acid residues are added employing standard peptide synthesismethodology to give the peptide dimer 6 (see FIG. 19C).

Peptide dimers, where each peptide has the sequence Asp Ala His Lys [SEQID NO:1] and where L is a glyceryl ester, can be synthesized as follows.The 3-substituted propane-1,2-diols of formula 7 in FIG. 19D, wherein Ris an alkyl or aryl, are commercially available. A lysine diester 8,wherein R is methyl, can be prepared as follows (see FIG. 19D). To astirred solution of Fmoc-D-Lys(Boc)-OH (20 mmole) in dry toluene (100mL; dry argon flushed) is added 3-methoxypropane-1,2-diol (200 mmole)and imidazole (15 mmole). The solution is stirred for 36 hours at roomtemperature. The solvent is removed in vacuo, and the residue isdissolved in ethyl acetate. This solution is washed with citric acidsolution (2%), water, 0.5 N NaHCO₃ solution, and again with water; thenthe organic layer is dried over magnesium sulphate (removal of thesolvent gives a pale yellow residue). The bis-protected lysine 8 isisolated by flash chromatography over silica and elution with mixturesof ethyl acetate/methanol. The peptide dimer 9 is then prepared from theprotected lysine dimer 8 employing classical peptide synthesismethodology (see FIG. 19D).

The physiologically-acceptable salts of the metal-binding compounds arealso included in the invention. Physiologically-acceptable salts includeconventional non-toxic salts, such as salts derived from inorganic acids(such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, andthe like), organic acids (such as acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, glutamic, benzoic, salicylic,and the like) or bases (such as the hydroxide, carbonate or bicarbonateof a pharmaceutically-acceptable metal cation). The salts are preparedin a conventional manner, e.g., by neutralizing the free base form ofthe compound with an acid.

A metal-binding compound of the invention can be used to reduce thedamage done by ROS or to reduce the metal ion concentration in an animalin need thereof. To do so, a metal-binding compound of the invention isadministered to the animal. Preferably, the animal is a mammal, such asa rabbit, goat, dog, cat, horse or human. Effective dosage forms, modesof administration and dosage amounts for the various compounds of theinvention may be determined empirically, and making such determinationsis within the skill of the art. It has been found that an effectivedosage is from about 2 to about 200 mg/kg, preferably from about 10 toabout 40 mg/kg, most preferably about 20 mg/kg. However, it isunderstood by those skilled in the art that the dosage amount will varywith the particular metal-binding compound employed, the disease orcondition to be treated, the severity of the disease or condition, theroute(s) of administration, the rate of excretion of the compound, theduration of the treatment, the identify of any other drugs beingadministered to the animal, the age, size and species of the animal, andlike factors known in the medical and veterinary arts. In general, asuitable daily dose of a compound of the present invention will be thatamount of the compound which is the lowest dose effective to produce atherapeutic effect. However, the daily dosage will be determined by anattending physician or veterinarian within the scope of sound medicaljudgment. If desired, the effective daily dose may be administered astwo, three, four, five, six or more sub-doses, administered separatelyat appropriate intervals throughout the day. Administration of thecompound should be continued until an acceptable response is achieved.

The compounds of the present invention may be administered to an animalpatient for therapy by any suitable route of administration, includingorally, nasally, rectally, vaginally, parenterally (e.g., intravenously,intraspinally, intraperitoneally, subcutaneously, or intramuscularly),intracisternally, transdermally, transmucosally, intracranially,intracerebrally, and topically (including buccally and sublingually).The preferred routes of administration are orally, intravenously, andtopically.

While it is possible for a metal-binding compound of the presentinvention to be administered alone, it is preferable to administer thecompound as a pharmaceutical formulation (composition). Thepharmaceutical compositions of the invention comprise a metal-bindingcompound or compounds of the invention as an active ingredient inadmixture with one or more pharmaceutically-acceptable carriers and,optionally, with one or more other compounds, drugs or other materials.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to theanimal. Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington's Pharmaceutical Sciences.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonosterate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugars, as well as high molecular weight polyethylene glycols and thelike.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound(s), may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical, transdermal or transmucosal administrationof a compound of this invention include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches, drops and inhalants.The active compound(s) may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to acompound or compound(s) of this invention, excipients, such as animaland vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound or compoundsof this invention, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder or mixturesof these substances. Sprays can additionally contain customarypropellants such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

The active ingredient (i.e., a metal-binding compound or compounds ofthe invention) may also be delivered through the skin using conventionaltransdermal drug delivery systems, i.e., transdermal patches, whereinthe active ingredient is typically contained within a laminatedstructure that serves as a drug delivery device to be affixed to theskin. In such a structure, the active ingredient is typically containedin a layer, or “reservoir,” underlying an upper backing layer. Thelaminated device may contain a single reservoir, or it may containmultiple reservoirs. In one embodiment, the reservoir comprises apolymeric matrix of a pharmaceutically acceptable contact adhesivematerial that serves to affix the system to the skin during drugdelivery. Examples of suitable skin contact adhesive materials include,but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes,polyacrylates, polyurethanes, and the like. Alternatively, thedrug-containing reservoir and skin contact adhesive are present asseparate and distinct layers, with the adhesive underlying the reservoirwhich, in this case, may be either a polymeric matrix as describedabove, or it may be a liquid or hydrogel reservoir, or may take someother form.

The backing layer in these laminates, which serves as the upper surfaceof the device, functions as the primary structural element of thelaminated structure and provides the device with much of itsflexibility. The material selected for the backing material should beselected so that it is substantially impermeable to the activeingredient and any other materials that are present. The backing layermay be either occlusive or nonocclusive, depending on whether it isdesired that the skin become hydrated during drug delivery. The backingis preferably made of a sheet or film of a preferably flexibleelastomeric material. Examples of polymers that are suitable for thebacking layer include polyethylene, polypropylene, polyesters, and thelike.

During storage and prior to use, the laminated structure includes arelease liner. Immediately prior to use, this layer is removed from thedevice to expose the basal surface thereof, either the drug reservoir ora separate contact adhesive layer, so that the system may be affixed tothe skin. The release liner should be made from a drug/vehicleimpermeable material.

Transdermal drug delivery devices may be fabricated using conventionaltechniques, known in the art, for example by casting a fluid admixtureof adhesive, active ingredient and vehicle onto the backing layer,followed by lamination of the release liner. Similarly, the adhesivemixture may be cast onto the release liner, followed by lamination ofthe backing layer. Alternatively, the drug reservoir may be prepared inthe absence of active ingredient or excipient, and then loaded by“soaking” in a drug/vehicle mixture.

The laminated transdermal drug delivery systems may, in addition,contain a skin permeation enhancer. That is, because the inherentpermeability of the skin to some active ingredients may be too low toallow therapeutic levels of the drug to pass through a reasonably sizedarea of unbroken skin, it is necessary to coadminister a skin permeationenhancer with such drugs. Suitable enhancers are well known in the art.

The pharmaceutical compositions of the invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, propellants such as fluorocarbons or nitrogen, and/orother conventional solubilizing or dispersing agents.

Preferred formulations for topical drug delivery are ointments andcreams. Ointments are semisolid preparations which are typically basedon petrolatum or other petroleum derivatives. Creams containing theselected active agent, are, as known in the art, viscous liquid orsemisolid emulsions, either oil-in-water or water-in-oil. Cream basesare water-washable, and contain an oil phase, an emulsifier and anaqueous phase. The oil phase, also sometimes called the “internal”phase, is generally comprised of petrolatum and a fatty alcohol such ascetyl or stearyl alcohol; the aqueous phase usually, although notnecessarily, exceeds the oil phase in volume, and generally contains ahumectant. The emulsifier in a cream formulation is generally anonionic, anionic, cationic or amphoteric surfactant. The specificointment or cream base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery.As with other carriers or vehicles, an ointment base should be inert,stable, nonirritating and nonsensitizing.

Formulations for buccal administration include tablets, lozenges, gelsand the like. Alternatively, buccal administration can be effected usinga transmucosal delivery system as known to those skilled in the art.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

As noted above, ROS have been reported to play a major role in a varietyof diseases and conditions. See Manso, Rev. Port. Cardiol., 11, 997-999(1992); Florence, Aust. N Z J. Opthalmol., 23, 3-7 (1992); Stohs, J.Basic Clin. Physiol. Pharmacol., 6, 205-228 (1995); Knight, Ann. Clin.Lab. Sci., 25, 111-121 (1995); Kerr et al., Heart & Lung, 25, 200-209(1996). Diseases and conditions involving or caused by excess metalions, and diseases and conditions wherein a reduction in theconcentration of metal ions would be desirable, are also known. Themetal-binding compounds of the invention can be used to treat any ofthese diseases and conditions and other diseases and conditions in whichROS or metal ions play a role by administering a metal-binding compoundof the invention as described above. “Treat” and variations thereof areused herein to mean to cure, prevent, ameliorate, alleviate, inhibit, orreduce the severity of a disease or condition or of at least some of thesymptoms or effects thereof.

Specific diseases and conditions that are treatable with themetal-binding compounds of the invention include adult respiratorydistress syndrome, aging, AIDS, angiogenic diseases, artherosclerosis(hypertension, senility and impotence), arthritis, asthma, autoimmunediseases, cancer (e.g., kidney, liver, colon, breast, gastrointestinaland brain), carcinogenesis, cellular damage caused by ionizing radiation(e.g., radiation of tumors), chronic granulomatous disease, cirrhosis,colitis, Crohn's disease, cystic fibrosis, degenerative diseases ofaging, diabetes (diabetic retinopathy, renal disease, impotence andperipheral vascular disease), eye diseases (e.g., cataracts, centralartery occlusion, benign monoclonal gammopathy, and maculardegeneration), emphysema, head injury and traumatic brain injury,hepatitis C, infertility (copper present in seminal fluid can damage orkill sperm and/or lower sperm motility), inflammation, inflammatorybowel disease, metastasis, ischemia, neoplastic diseases, neurologicaldiseases, neurological trauma, neurodegenerative diseases (e.g.,Alzheimer's disease, amyotropic lateral sclerosis, Huntington's chorea,Parkinson's disease, multiple sclerosis, and senile dementia),pancreatitis, peripheral vascular disease, prion disease (transmissiblespongiosiform encephalomyopathy), pulmonary embolism, renal disease(dialysis patients), reperfusion, scleroderma, sepsis, shock, tissuedamage occurring upon administration of chemotherapeutics, tissue damageafter surgery (e.g., transplantation surgery, open heart surgery, andany surgery where the blood supply to a tissue is cut off, and surgicalischemia of the limbs (tourniquet injury)), toxic reactions (e.g,herbicide poisoning, transition metal (copper, cobalt, and nickel)poisoning, carbon monoxide poisoning, and antibiotic toxicity),traumatic crush injury, and Wilson's disease (congenital high levels ofcopper).

Specific ischemic conditions and diseases treatable with themetal-binding compounds of the invention include:

Central nervous system ischemia—

-   -   Brain ischemia after surgery    -   Hyperthermia brain injury    -   Perinatal hypoxia-induced ischemia (“cerebral palsy”)    -   Seizures    -   Spinal cord injury    -   Stroke (thrombotic, embolic or hemorrhagic cerebrovascular        accident)    -   Transient ischemic attack    -   Traumatic brain injury

Cardiac ischemia—

-   -   Acute myocardial infarction    -   Angina pectoris    -   Arrythmias    -   Cardiac ischemia after surgery    -   Congestive heart failure    -   Myocardial “stunning” (low cardiac output syndrome)

Ischemic bowel disease

Placental ischemia and fetal distress

Pulmonary embolism

Surgery where the blood supply to a tissue or organ is cut off—

-   -   Angioplasty    -   Cardiac bypass surgery    -   Transplantation surgery (both the donor organ and the recipient        of the organ)

Surgical ischemia of the limbs (tourniquet injury).

An angiogenic disease or condition is a disease or condition involving,caused by, exacerbated by, or dependent on angiogenesis. Angiogenesis isthe process of new blood vessel formation in the body. Copper isrequired for angiogenesis. See PCT application WO 00/21941 and “The RoleOf Copper In The Angiogenesis Process(http://www.cancerprotocol.com/role_of_copper.html, Jan. 28, 2002), andreferences cited in both of them. In particular, copper is involved inthe activation of growth factors (such as the dimerization of b-FGF andserum Cu²⁺-GHK), activation of angiogenic factors (such as Cu²⁺-(K)GHKderived from SPARC), cross-linking of the transitional matrix (e.g.,collagens VIII and I by Cu²⁺-dependent lysyl oxidase), and formation ofbasement membrane (e.g., collagens IV and elastin by Cu²⁺-dependentlysyl oxidase).

Specific angiogenic diseases and conditions treatable with themetal-binding compounds of the invention include neoplastic diseases(e.g., tumors (e.g., tumors of the bladder, brain, breast, cervix,colon, rectum, kidney, lung, ovary, pancreas, prostate, stomach anduterus) and tumor metastasis), benign tumors (e.g., hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyrogenic granulomas),hypertrophy (e.g., cardiac hypertrophy induced by thyroid hormone),connective tissue disorders (e.g., rheumatoid arthritis andatherosclerosis), psoriasis, ocular angiogenic diseases (e.g., diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia, andrubeosis), cardiovascular diseases, cerebral vascular diseases,endometriosis, polyposis, obesity, diabetes-associated diseases,hemophiliac joints, and immune disorders (e.g., chronic inflammation andautoimmunity). The metal-binding compounds of the invention can also beused to inhibit the vascularization required for embryo implantation,thereby providing a method of birth control.

High copper levels have been found in the serum and tumors of patientswith many types of progressive tumors. As noted above, copper plays amajor role in angiogenesis, thereby contributing to tumor growth andmetastasis. Also, metals, particularly copper and nickel, have beenreported to be carcinogens. Thus, the metal-binding compounds of theinvention may be used to reduce copper levels in cancer patients. Themetal-binding compounds of the invention may also be used to inhibit(reduce or prevent) carcinogenesis in individuals at risk (e.g.,metal-exposed individuals, such as welders, machinists, autobodyrepairmen, etc.).

Specific inflammatory diseases and conditions treatable with themetal-binding compounds of the invention include acute respiratorydistress syndrome, allergies, arthritis, asthma, autoimmune diseases,bronchitis, cancer, Crohn's disease, cystic fibrosis, emphysema,endocarditis, gastritis, inflammatory bowel disease, ischemiareperfusion, multiple organ dysfunction syndrome, nephritis,pancreatitis, respiratory viral infections, sepsis, shock, ulcerativecolitis and other inflammatory disorders.

Acidosis is present in, or plays a role in, a number of diseases andconditions, including hypoventilation, hypoxia, ischemia, prolonged lackof oxygen, severe dehydration, diarrhea, vomiting, starvation, AIDS,sepsis, kidney disease, liver disease, metabolic diseases (e.g.,advanced stages of diabetes mellitus), and neurodegenerative diseases(e.g., Alzheimer's). Acidosis is also caused by certain medications(e.g., large amounts of aspirin and oral medications used to treatdiabetes), and instances of mild acidosis have been reported to increasewith age (Knight, “Metal Heads,” New Scientist, Aug. 26, 2000,http://www.purdeyenvironment.com/Full%20New%20scientist001.htm). Undernormal conditions, copper is bound to plasma proteins and peptides,primarily ceruloplasmin and albumin. In acidotic conditions, copper isreleased from the proteins to which it is normally bound. It isestimated that 40-70% of weakly bound copper is released at pH 6.0. Freecopper can participate in reactions that lead to the formation of ROSand causes a number of other deleterious effects, such as interferingwith metabolism and energy ultilization. Thus, the metal-bindingcompounds of the invention may be used to treat diseases or conditionsinvolving acidosis to prevent damage due to ROS, to prevent otherdeleterious effects of free copper, or both.

Sepsis can also be treated using the metal-binding compounds of theinvention. Sepsis is a systemic inflammatory response to infection.Sepsis is also characterized by ischemia (caused by coagulopathy andsuppressed fibrinolysis) and acidosis.

A compound of the invention is preferably administered prophylactically.For instance, a compound of the invention is preferably administeredprior to and/or simultaneously with reperfusion of an ischemic tissue ororgan (e.g., prior to and/or simultaneously with angioplasty ortreatment with clot dissolving drugs, such as tissue plasminogenactivators). Of course administration of a compound of the inventionshould be continued for a period of time after reperfusion has beenachieved. Similarly, a compound of the invention should be administeredprior to and/or during surgery (e.g., open-heart surgery or surgery totransplant an organ into an animal), and administration of the compoundshould be continued for a period of time after the surgery. As anotherexample of prophylactic administration, a compound of the invention canbe administered to a patient presenting symptoms of a serious condition(e.g., cerebrovascular ischemia or cardiovascular ischemia) while thepatient is tested to diagnose the condition. In this way, the patientwill be protected during the time it takes to diagnose such conditions,and treatment with the metal-binding compounds of the invention may alsoprolong the time during which other therapies (e.g., administration oftissue plasminogen activator for cerebrovascular ischemia) can beadministered. As yet a further example, a compound of the invention canbe administered at the time a patient is to undergo radiation therapy(e.g., radiation for a tumor or prior to a bone marrow transplant).

A compound of the invention can also be used to treat patients who havesuffered blunt trauma. In particular, a compound of the presentinvention may be very beneficial in treating patients suffering frommultiple blunt trauma who have a low albumin level, since it has beenfound that a low albumin level is a predictor of mortality in suchpatients. More specifically, 34 patients suffering from multiple blunttrauma were studied. These patients were admitted to the intensive careunit of Swedish Hospital, Denver, Colo. in 1998. Two groups of patientswere matched by a trauma surgeon by age, injury severity score (ISS),and type and area of injury without knowledge of the albumin levels ofthe patients. One group was composed of the patients who died, and theother group was composed of survivors. Following the match, theadmission albumin levels were retrieved from the medical records by anindependent observer, and the albumin levels of the two groups werecompared. For the 17 survivors, the mean albumin level was 3.50±1.00g/dl. For the 17 patients who died, the mean albumin level was 2.52±0.73g/dl. The % variance was 28.6 and 28.9, respectively, and the p-valuewas 0.0026 (95% confidence interval 0.3462-0.4771).

The compounds of the invention may be given alone to reduce the damagedone by ROS. Alternatively, the compounds of the invention can be givenin combination with “free radical scavengers.” “Free radical scavengers”include superoxide dismutase, catalase, glutathione peroxidase, ebselen,glutathione, cysteine, N-acetyl cysteine, penicillamine, allopurinol,oxypurinol, ascorbic acid, α-tocopherol, Trolox (water-solubleα-tocopherol), β-carotene, fatty-acid binding protein, fenozan,probucol, cyanidanol-3, dimercaptopropanol, indapamide, emoxipine,dimethyl sulfoxide, and others. See, e.g., Das et al., Methods Enzymol.,233, 601-610 (1994); Stohs, J. Basic Clin. Physiol. Pharmacol., 6,205-228 (1995). The compounds of the invention can also be given incombination with another metal-binding peptide or non-peptide chelator(suitable metal-binding peptides and non-peptide chelators are describedabove and others are known in the art). For instance, a peptide P₁(i.e., peptide P₁-P₂ wherein n=0 in the formula of P₂), which bindsCu(II) tightly, could be given in combination with a separate peptidesuitable for binding Cu(I) (suitable Cu(I)-binding peptides aredescribed above). As another example, a peptide P₁ could be given incombination with a separate peptide or non-peptide chelator capable ofbinding iron. Suitable iron-binding peptides and non-peptide chelatorsare described above and others are known in the art (e.g., deferoxaminemesylate). The compounds of the invention can, of course, also be givenalong with standard therapies for a given conditions (e.g., insulin totreat diabetes).

The metal-binding compounds of the invention can also be used to reducethe damage done by ROS to a cell, a tissue or organ that has beenremoved from an animal. To reduce the damage done by ROS to a tissue oran organ, the tissue or organ is contacted with a solution containing aneffective amount of a metal-binding compound of the invention. Manysuitable solutions are known. See, e.g., Dunphy et al., Am. J. Physiol.,276, H1591-H1598 (1999); Suzer et al., Pharmacol. Res., 37, 97-101(1998); Hisatomi et al., Transplantation, 52, 754-755 (1991); U.S. Pat.No. 5,710,172. Effective amounts of the metal-binding compound toinclude in such solutions can be determined empirically, and doing so iswithin the skill in the art. The harvested tissue or organ maysubsequently be used for transplantation into a recipient or forresearch purposes (e.g., using a perfused liver to screen drugs). Themetal-binding compounds of the invention can be used alone or can beused in combination with a free radical scavenger or anothermetal-binding compound (see above).

Cells isolated from an animal can be stored or cultured in a mediumcontaining an effective amount of a metal-binding compound of theinvention. Many suitable media are known. Effective amounts of themetal-binding compound to include in the medium can be determinedempirically, and doing so is within the skill in the art. The cells maybe administered to a recipient in need thereof (e.g., for cellularimmunotherapy or gene therapy) or may be used for research purposes.

In addition, media containing an effective amount of a metal-bindingcompound of the invention can be used in in vitro fertilization (IVF)procedures for reducing the damage done by ROS to gametes (sperm and/orova), zygotes, and blastocysts during collection, storage and/orculture. In particular, seminal fluid is known to contain substantialamounts of copper and fructose, conditions suitable for the productionof ROS. Many suitable media for use in IVF procedures are known (e.g.,Gardner media G1, G2, etc.). Effective amounts of the metal-bindingcompound to include in the media can be determined empirically, anddoing so is within the skill in the art.

The invention further provides a kit for reducing the damage done by ROSto a cell, a tissue or organ that has been removed from an animal. Thekit is a packaged combination of one or more containers holding reagentsand other items useful for preserving harvested cells, tissues ororgans. The kit comprises a container holding a metal-binding compoundof the invention. Suitable containers include bottles, bags, vials, testtubes, syringes, and other containers known in the art The kit may alsocontain other items which are known in the art and which may bedesirable from a commercial and user standpoint, such as a container forthe cells, tissue or organ, diluents, buffers, empty syringes, tubing,gauze pads, disinfectant solution, etc.

It is to be noted that “a” or “an” entity refers to one or more of thatentity. For example, “a cell” refers to one or more cells.

EXAMPLES Example 1 Synthesis of Tetrapeptide Asp Ala His Lys [SEQ IDNO:1]

This example describes the synthesis of the tetrapeptide Asp Ala His Lys[SEQ ID NO:1] composed of all L-amino acids using standard solid-phasesynthesis techniques. First, 9-fluorenylmethyloxycarbonyl(Fmoc)-protected Asp (ν COO— ester; Tolsulfonyl) on Wang resin (0.6mmole; Nova Biochem) was suspended in a solution ofpiperidine/dimethylformamide (DMF) (40% v/v; 3 ml) for 30 min withoccasional agitation. At the end of this period, the solvent wasdrained, and the resin was washed sequentially with DMF anddichloromethane (DCM; 5×3 ml). A ninhydrin test was used to monitor thereaction. The resin was swollen with DMF (˜1 ml). The C-protectedt-benzyloxycarbonyl (Boc) ester of alanine in DMF was added, followed bya mixture of diisopropylamine (8 equivalent) and2-(1H-benzotriazole-1-yl)-1,2,3,3-tetramethyluroniumtetrafluoroborate(TBTU-) (4 equivalents). The resin was shaken for about 24 hours, andthe reaction was monitored by the ninhydrin test. At the end of thisperiod, DMF was drained, and the resin was washed with DMF and DCM. Thesolution was drained, and the beads were washed with DCM (3×2 ml). Theprotecting group of the dipeptide-resin was removed, and the beads weresuspended in DMF. Amino protected (benzyloxy) derivative of histidine (4mmole) was added, followed by mixture of diisopropylamine (8 equivalent)and TBTU-(4 equivalent). The resin was shaken for about 24 hours, andthe reaction monitored by ninhydrin test. At the end of this period, DMFwas drained, and the resin was washed with DMF and DCM. Thetripeptide-resin was briefly dried in a gentle stream of nitrogen andsuspended in nitrogen-saturated DMF. Protected lysine was added,followed by a mixture of diisopropylamine (8 equivalent) and TBTU-(4equivalent). The resin was shaken for about 24 hours, and the reactionmonitored by the ninhydrin test. At the end of this period, DMF wasdrained and the resin was washed with DMF and DCM. The Boc protectinggroup was carefully removed to give the tetrapeptide bound to the resin,with a typical loading of 5 mmole/g. The resin bound tetrapeptide (0.25gm; 5 mmolar) was treated with trifluoroacetic acid (TFA) and was shakenfor 24 hours. At the end of this period, the ninhydrin test gave a bluecolor, indicating the release of the tetrapeptide from the resin. Insome circumstances, addition of 5% (V/V) of DMF to TFA accelerated therate of release of the peptide from the resin. Removal of TFA at reducedpressure gave the tetrapeptide (all D) as TFA salt and was dried undervacuum at 5° C. for 24 hours. The residue was a white powder and wascharacterized by spectrometric methods.

A number of enantiomers of the tetrapeptide can be prepared in thismanner. For example, use of D-amino acids in the peptide synthesis formsthe tetrapeptide containing all D-amino acids. Also, combinations ofL-amino acids and D-amino acids can be used.

Example 2 Preparation of Cyclohexanediamine Derivative of Asp Ala HisLys [SEQ ID NO:1]

Trans-diaminocyclohexane was prepared by resolving cis/trans1,2-diaminocyclohexane (Aldrich-Sigma) as the tartaric acid salt. TheR-trans isomer melts at 75° C. and the S-trans isomer melts between43-45° C. (Ph.D. Thesis, P. D. Newman, University College, Cardiff,U.K., 1994). The trans-diaminocyclohexane (10 gm) was then suspended inanhydrous toluene (30 mL) and cooled to 5° C. in an ice bath, andbromoacetic acid (8 gm) in toluene (25 mL) was added dropwise. At theend of the addition, the reaction temperature was raised to 30° C. andkept at that temperature for a further 5 hours. Toluene was evaporated,and the R-trans 1,2-diaminocyclohexane diacetic acid was crystallizedfrom hexane/toluene to give a white solid (yield 70%). The product wascharacterized by spectroscopic methods.

The resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspendedin DMF (5 mL) and was treated with the R-trans1,2-diaminocyclohexanediacetic acid (20 mg) followed by addition of amixture of diisopropylamine (8 equivalent) and TBTU-(4 equivalent). Theresin was shaken for about 24 h on a roller. Then, the resin was washedwith DMF followed by DCM (5×3 mL) and partially dried. Hydrolysis of theresin linkage was effected by treating the resin-bound reaction productwith TFA (5 mL; 5 hr). The resin was separated and washed with DCM. Thewashings were combined with TFA and concentrated under vacuum. Theresidue (cyclohexanediamine tetrapeptide; formula given in FIG. 3D whereR₅ is H) was characterized by spectrometric analysis.

Example 3 Preparation of Tetrapeptide Tetracetic Acid

The resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspendedin DMF (5 mL) and treated with excess (10-fold) chloroacetic acid. Theresin was shaken at room temperature for 48 hours, followed by heatingto 60° C. for a further hour. DMF was removed by filtration, and theresin was washed with DMF followed by DCM (5×3 mL). Partially driedresin was used without further treatment in the next stage. Hydrolysisof the resin linkage was effected by treating the resin-bound reactionproduct with TFA (5 mL; 5 hr). The resin was separated and washed withDCM. The washings were combined with TFA and concentrated under vacuum(yield 30%). The product (formula given in FIG. 4) was characterized byspectrometric methods.

Example 4 Preparation of Mesoporphyrin IX Tetrapeptide

The resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspendedin DMF (5 mL) and treated with mesoporphyrin IX dicarboxylic acid (10μmole; formula given in FIG. 6A), followed by addition of a mixture ofdiisopropylamine (8 equivalent) and TBTU-(4 equivalent). The resin wasshaken for about 24 hours on a roller kept in a dark chamber. The resinwas washed with DMF followed by DCM (5×3 mL) and partially dried.Hydrolysis of the resin linkage was effected by treating the resin-boundreaction product with TFA (5 mL; 5 hr). The resin was separated andwashed with DCM/TFA mixture (1:1.5 mL). The washings were combined andconcentrated under vacuum. The porphyrin tetrapeptide (formula given inFIG. 6C) was purified by semi-preparative HPLC (yield 60%). Thestructure was confirmed by spectrometric methods.

This procedure can be used to synthesize other porphyrin-peptides, suchas mesoporphyrin I and related molecules.

Example 5 Preparation of Tetrabispiridylethyl Tetrapeptide

The resin-bound tetrapeptide prepared in Example 1 (20 mg) was suspendedin DMF (5 mL) and treated with bromoethylpyridine (20 μmole). This wasfollowed by the addition of pyridine (0.5 mL). The resin was shaken forabout 48 hours on a roller. The resin was washed with DMF, followed byDCM (5×3 mL) to remove all of the unreacted monomers, and then driedunder vacuum for 30 minutes. Hydrolysis of the resin linkage waseffected by treating the resin-bound reaction product with TFA (5 mL; 5hr). The resin was separated and washed with DCM/TFA mixture (1:1.5 mL).The washings were combined and concentrated under vacuum. Thepyridylethyl tetrapeptide derivative (formula given in FIG. 5) waspurified by semi-preparative HPLC (yield 50%). The structure wasconfirmed by spectrometric methods.

This procedure can be applied to other heterocycles, such asphenanthroline and related molecules.

Example 6 Preparation of Aryl Derivative of Asp Ala His Lys [SEQ IDNO:1]

A derivative having the formula shown in FIG. 1B, wherein R₁ is phenyl,was prepared. Diethylacetamidomalonate (10 gm) in anhydrous ethanol (100mL) was added to a slurry of sodium ethoxide in ethanol (5 gm; 50 mL)and heated to reflux for 30 min. The product was cooled (10° C.) andreacted with ethyl α-bromophenyl acetate (5 gm). The reaction wasallowed to proceed to completion (24 h), and excess sodium ethoxide wasneutralized with dilute acid. The triester was extracted intoethylacetate and, upon removal of solvent, gave a viscous liquid. Thecrude product was hydrolyzed with hydrochloric acid (100 mL) anddecarboxylated to give phenyl substituted aspartic acid (10 gm). TheN-benzoyloxy t-butyl derivative was prepared using a standard reactionsequence. To the resin-bound tripeptide (Lys His Ala) prepared asdescribed in Example 1 (20 mg) in DMF was added the N-benzoyloxy-t-butylaspartic acid derivative, followed by a mixture of diisopropylamine (8equivalent) and TBTU-(4 equivalent). The resin was shaken for about 24h, and the reaction monitored by the ninhydrin test. At the end of thisperiod, DMF was drained, and the resin was washed with DMF and DCM. Thesolution was drained, and the beads washed with DCM (3×2 ml). Thetetrapeptide derivative was isolated by careful hydrolysis.Stereoisomers of the tetrapeptide were separated by preparative-scaleHPLC.

Example 7 Inhibition of the Generation of ROS By The Tetrapeptide AspAla His Lys [SEQ ID NO:1]

A tetrapeptide having the sequence L-Asp L-Ala L-His L-Lys [SEQ ID NO:1](the L-tetrapeptide) was obtained from one or more companies thatprovide custom synthesis of peptides, including Ansynth Services, QCB,Genosys and Bowman Research. The peptide was prepared by standard solidphase synthesis methods (see also Example 1).

The ability of the L-tetrapeptide to inhibit the generation of ROS wastested as described in Gutteridge and Wilkins, Biochim. Biophys. Acta,759, 38-41 (1983) and Cheeseman et al., Biochem. J., 252, 649-653(1988). Briefly, Cu(II) and H₂O₂ were mixed causing the generation ofhydroxyl radicals in a Fenton-type reaction. The hydroxyl radicalsattack the sugar 2-deoxy-D-ribose (the sugar residue of DNA) to producefragments. Heating the fragments at low pH produces malonaldehyde that,upon the addition of 2-thiobarbituric acid, yields a pink chromogenwhich can be measured spectrophotometrically at 532 nm. Thus, theabsorbance at 532 nm is a measure of the damage to 2-deoxy-D-ribose.

The assay was performed with and without the L-tetrapeptide. The resultsare summarized in Table 1. As can be seen from Table 1, when theL-tetrapeptide was present at Cu(II):tetrapeptide ratios of 1:1.2 and1:2, the degradation of 2-deoxy-D-ribose was inhibited by 38% and 73%,respectively. Clearly, the L-tetrapeptide inhibited the degradation of2-deoxy-D-ribose by hydroxyl radicals.

TABLE 1 CuCl₂ H₂O₂ Tetra-peptide OD at Percent (mM) (mM) (mM) 532 nmInhibition Control 0.1 2.0 0.0 0.124 Tetrapeptide 0.1 2.0 0.12 0.077 38Control 0.1 2.0 0.0 0.175 Tetrapeptide 0.1 2.0 0.2 0.048 73

A similar assay was also performed using a tetrapeptide having thesequence Asp Ala His Lys composed of all D-amino acids (D-tetrapeptide).The D-tetrapeptide was obtained from one or more companies that providecustom synthesis of peptides, including Ansynth Services and QCB. Thepeptide was prepared by standard solid phase synthesis methods (seeExample 1)

The ability of the D-tetrapeptide to inhibit the generation of ROS wastested as described by Zhao and Jung, Free Radic Res, 23(3), 229-43(1995). Briefly, Cu(II) and ascorbic acid were mixed causing thegeneration of hydroxyl radicals in a Fenton-type reaction. The advantageof using ascorbic acid instead of hydrogen peroxide is that ascorbicacid does not interfere with other assays (i.e. LDH assay) which is notthe case with peroxide. The hydroxyl radicals attack the sugar2-deoxy-D-ribose to produce fragments. Heating the fragments at low pHproduces malonaldehyde that, upon the addition of 2-thiobarbituric acid,yields a pink chromogen which can be measured spectrophotometrically at532 nm. Thus, the absorbance at 532 nm is a measure of the damage to2-deoxy-D-ribose.

Establishing optimal Cu(II) and ascorbic acid concentrations was thefirst step in developing this protocol. First, a constant Cupconcentration of 10 μM was used based on this level being thephysiological concentration found in the body (bound and unboundCu(II)). The ascorbic acid concentrations were varied in order toestablish a linear range. The ascorbic acid concentration chosen was 500μM since it gave the most absorbance at 532 nm and still fell in thelinear range. Interestingly, at ascorbic acid concentrations greaterthan 500 μM, there was a steady decrease in hydroxyl radicals presumablydue to ascorbic acid's dual effect as a hydroxyl radical generator atlow concentrations and an antioxidant at high concentrations.

Using the aforementioned concentrations for Cu(II) and ascorbic acid, atitration curve was established for the D-tetrapeptide. Briefly, theD-tetrapeptide was pre-incubated with Cu(II) for 15 minutes at roomtemperature prior to adding ascorbic acid. This was done to permit theD-tetrapeptide to bind with the Cu(II) and therefore inhibit ROSgeneration. As can be seen from the table, when theCu(II):D-tetrapeptide ratio was between 4:1 to 4:7, there was little tono inhibition of hydroxyl radical generation. When the ratio was 1:2 orhigher, there was total inhibition of hydroxyl radical production.

TABLE 2 Cu(II): Cu(II) Ascorbic D-Tetrapeptide % Inhi- D-Tetrapeptide(μM) Acid (μM) (μM) A532 bition 1:0 10 500 0 0.767 4:1 10 500 2.5 0.7512:1 10 500 5 0.743 1:1 10 500 10 0.751 4:5 10 500 12.5 0.789 2:3 10 50015 0.774 4:7 10 500 17.5 0.737 1:2 10 500 20 0.029 96.2 1:4 10 500 400.016 97.9

Example 8 Testing of Asp Ala His Lys D-Tetrapeptide In A LangendorffReperfusion Model

Blood-perfused hearts were prepared essentially as in previous studies(Galiñanes et al., Circulation, 88:673-683 (1993); Kolocassides et al.,Am. J. Physiol., 269:H1415-H1420 (1995); Hearse et al., J. Mol. Cell.Cardiol., 31:1961-1973 (1999)). See FIG. 8. The procedures are describedbriefly below.

Male Wistar rats, obtained from Bantin and Kingman Universal, UK, wereused. All animals received humane care in compliance with the“Principles of Laboratory Animal Care” formulated by the NationalSociety for Medical Research and the “Guide for the Care and Use ofLaboratory Animals” prepared by the National Academy of Sciences, andpublished by the US National Institutes of Health (NIH Publication No.85-23, revised 1996).

Support rats (300-400 g) were anesthetized with sodium pentobarbitone(60 mg/kg, intraperitoneally) and anticoagulated with heparin (1000IU/kg intravenously). The right femoral vein and left femoral arterywere exposed by blunt dissection and cannulated (18G and 22G Abbocath-Tcatheters respectively) for the return and supply of blood to theperfused heart. An extracorporeal circuit was established, primed withGelofusine® plasma substitute (B. Braun Medical Ltd., Aylesbury, UK) andwas maintained for 15 minutes (min) before connection to the isolatedheart. This period was to ensure that the priming solution wasadequately mixed with the blood of the support rat and that the entirepreparation was stable. Each 500 ml of Gelofusine® contains 20.00 gsuccinated gelatin (average molecular weight 30,000), 3.65 g. sodiumchloride, water for injection to 500 ml (electrolytes mmol/500 ml:cations Na 77, anions Cl 62.5, pH 7.4). Prior to perfusing the donorheart, an additional 7-8 ml of blood from a rat of the same strain wasadded to the central reservoir. This was to ensure that the support rathad an adequate supply of blood during the experiment when blood was notrecirculated but instead collected for a 2 min period. A peristalticpump (Gilson Minipuls 3) was located on the arterial outflow of thesupport rat and flow through the extracorporeal circuit was increasedgradually over 10 min to a value of 2.5 ml/min. This gradual increaseprevented the drop in arterial pressure that would have occurred if aflow rate of 2.5 ml/min had been established immediately. The blood waspumped through a cannula (to which the aorta of the perfused heart wouldsubsequently be attached) and returned, by gravity, via a reservoir andfilter to the venous inflow line of the support animal. An air-filledsyringe above the perfusion cannula acted as a compliance chamber, whichserved to dampen oscillations in perfusion pressure which occurred as aconsequence of the contraction of the isolated heart and the peristalticaction of the pump. The support animal was allowed to breathe a mixtureof 95% O₂+5% CO₂ through a 35% Venturi face mask. The flow rate wasadjusted to maintain blood pO₂ and pCO₂ within the physiological range.Body temperature was stabilized at 37.0 (±0.5)° C. by means of athermostatically-controlled heating pad and was monitored by a rectalthermometer. Blood pressure was monitored by means of a pressuretransducer attached to the arterial line. All pressure transducers wereconnected to a MacLab (ADInstruments, Australia), which was runcontinuously through the experiment. Blood gas (pO₂, pCO₂, pH),hematocrit, glucose and electrolyte levels (Na⁺, K⁺, Ca⁺) of the supportrat were monitored before the donor heart was attached to theextracorporeal circuit and at the end of each experiment. During thecourse of the experiment, minimum amounts of donor blood (from anotherat of the same strain) were transfused as required so as to maintain thevolume and stability of the preparation. Additional heparin andpentobarbitone were administered into the central reservoir as required.

To isolate hearts, each rat (270-350 g) was anesthetized with diethylether and anticoagulated with heparin (1000 IU/kg intravenously). Theheart was then immediately excised and immersed in cold (4° C.)Gelofusine®. The aorta was rapidly cannulated and perfused in theLangendorff mode, (Langendorff, Pflugers Archives fur die GestamtePhysiologie des Menschen and der Tiere, 61:291-332 (1895)) with arterialblood from the support animal, at a constant flow rate of 2.5 ml/min.After removal of the left atrial appendage, a fluid-filled ballooncatheter (for the measurements of left ventricular systolic anddiastolic pressures, and, by difference, left ventricular developedpressure), attached to a pressure transducer, was introduced into theleft ventricle via the mitral valve. The balloon was inflated with wateruntil a left ventricular end diastolic pressure (LVEDP) of between 4-8mmHg was obtained. Heart rate was calculated from the pressure trace andexpressed as beats per minute (bpm). Perfusion pressure was measured viaa sidearm of the aortic cannula. All pressure transducers were connectedto a MacLab, which was run continuously through the experiment.

Excised hearts were randomly assigned to two treatment groups (see FIG.9; 6 hearts/group) and aerobically perfused for 20 min prior to (i)saline control with 2 min saline infusion immediately prior to a 30 minperiod of ischemia plus 2 min saline infusion at the onset ofreperfusion and (ii) drug with 2 min drug infusion immediately prior toa 30 min period of ischemia (the drug was therefore trapped in thevasculature for the duration of the ensuing ischemic period), plus a 2min drug infusion at the onset of reperfusion. Hearts were thensubjected to 30 min of global, zero-flow ischemia, during which timethey were immersed in saline at 37.0° C. Ischemia was initiated byclamping the line leading from the pump to the aortic cannula, thusdiverting the flow away from the isolated heart back to the supportanimal, via the bypass line. Hearts were then reperfused for 40 min,during which time contractile function was continuously measured.

The drug, whose identity was unknown to the researchers performing theexperiments, was the tetrapeptide D-Asp D-Ala D-His D-Lys. Thetetrapeptide was supplied to the researchers by Bowman Research, UK,dissolved in saline at a concentration of 16.7 mg/mL. It was infused assupplied without any dilution or modification. Physiological saline wassupplied by Baxter, UK and used in controls. Fresh solutions of salineand the drug were used daily.

Drug or vehicle was infused into a sidearm of the aortic cannula bymeans of a peristaltic pump (Gilson Minipuls 3), set at a constant flowof 0.25 ml/min. Since blood flow through the aortic cannula was 2.5ml/min, and drug infusion was 0.25 ml/min, the final concentration ofdrug delivered to the heart was 1/11th of that supplied by BowmanResearch. During the 2 min period of pre-ischemic vehicle or druginfusion and at the time points indicated in FIG. 9 arterial and venousblood samples were collected, centrifuged and frozen for analysis. Theinfusion was then repeated for the first 2 min of the reperfusionperiod, during which time the blood was not collected, but recirculated.

Predefined exclusion criteria stated that: (i) support animals would beexcluded from the study if they did not attain a stable systolic bloodpressure ≧80 mm Hg before cannulation of the donor heart, (ii) donorhearts would be excluded from the study if, at the 20 min baselinepre-intervention reading, LVDP≦100 mm Hg or (iii) blood chemistry valueswere outside the normal range.

Results are expressed as mean±SEM. All recovery values are expressed asa percent of the pre-intervention baseline value (measured 20 min afterthe onset of the experiment) for each individual heart. The two-tailedunpaired Student's t test was used for the comparison of two meansbetween groups. A difference was considered statistically significantwhen p <0.05.

For reasons of quality control and to allow application of predefinedexclusion criteria, the stability and reproducibility of the system weremonitored by measuring the blood chemistry (pH, pO₂, pCO₂, haematocrit,Na⁺, K⁺ and Ca²⁺, glucose) and baseline contractile function of eachsupport animal (immediately before perfusing a donor heart and at theend of each experiment) and each perfused heart. Table 3 reveals thatthere were only minor changes in each index measured, confirming thatsimilar perfusion conditions applied in both study groups and that allvalues were within the acceptable physiological range. The systolicpressure and heart rate of the support rats are shown in Table 4. As canbe seen, there were no significant differences between the two studygroups at the 15 min baseline reading.

Table 5 shows that there were no significant differences between groupsat the end of the 20 min aerobic perfusion period (i.e. just prior tothe infusion of drug or vehicle) in LVDP, heart rate and perfusionpressure. Thus, for LVDP, the primary endpoint in the study, the meanvalues were 177.3±10.6 mmHg and 177.2±5.6 mmHg for the groups that wereto become saline control and drug treated.

As expected, myocardial ischemia caused cessation of myocardialcontraction, with cardiac arrest initially in the diastolic state.However, as ischemic injury developed with time an increase in diastolicstate occurred as the heart went into ischemic contracture. The temporalprofiles for the development of ischemic contracture in each of thestudy groups is shown in FIG. 10. As can be seen from Table 6, therewere no differences in any of the measured indices, although there issome evidence of a trend to delay time-to-50% contracture in the drugtreated group, which is suggestive with protection.

FIG. 11 shows the profiles for the mean recovery of LVDP (expressed as apercent of baseline pre-intervention values) in both study groups. It isevident that hearts in the saline control group recovered slowly andpoorly, such that by the end of the 40 min reperfusion period, LVDP wasonly 15.3±3.2% of the pre-intervention control. By contrast, hearts inthe drug group recovered more rapidly and to a greater extent(50.5±9.3%).

FIG. 12 shows the absolute values for the left ventricular end diastolicpressure in both study groups during the 40 min period of reperfusion.In both groups, the high levels of LVEDP resulting from the contracturewhich developed during ischaemia fell with time towards thepre-intervention control value. However, the drug group normalized theirLVEDP more quickly and more completely than that seen in the salinecontrol group, the difference being significant at every time pointstudied. This would be consistent with the enhanced recovery seen duringreperfusion in the drug group.

A comparison of the heart rates obtained in the saline control group anddrug, as shown in FIG. 13 reveals that these two groups were essentiallyidentical (115.0±3.8 versus 127.2±22.8%).

As shown in FIG. 14, the perfusion pressure during the 40 minreperfusion period was essentially constant in both groups and thevalues did not differ significantly between each other at the end of the40 min reperfusion period. In saline controls, the mean value at the endof reperfusion was 109.9±8.2% of its pre-intervention control, whilethat of the drug group was 87.4±8.0% of its pre-intervention value.Thus, although the values for drug tended to be lower throughout thereperfusion period (which is consistent with the observedcardioprotection), these changes did not reach statistical significance.

The results of this pilot study indicate that, in the isolatedblood-perfused rat heart, Asp Ala His Lys appears to have significantand substantial protective properties as assessed by an approximatelythree and a half (3.5) fold (15.3±3.2% to 50.5±9.3%) enhancement ofpost-ischemic functional recovery. The magnitude of protection is equalto some of the most powerful interventions studied.

TABLE 3 Composition of the blood perfusing the isolated donor heartsValue prior to attaching donor heart End of experiment Index SalineControl Drug* Saline Control Drug* pH 7.29 ± 0.01  7.26 ± 0.01  7.33 ±0.02  7.31 ± 0.02 pCO₂ (mmHg) 60.0 ± 3.2  67.7 ± 2.2 57.6 ± 4.9 65.6 ±4.7 pO₂ (mmHg) 230.6 ± 14.8  281.7 ± 23.2 241.0 ± 37.2 252.3 ± 33.9Haematocrit (%) 27.5 ± 0.9  27.3 ± 1.8 26.8 ± 1.7 27.0 ± 1.5 Na⁺(mmol/L) 146.7 ± 0.3  146.2 ± 0.6  145.7 ± 0.2  146.2 ± 0.5  K⁺ (mmol/L)3.3 ± 0.1  3.2 ± 0.1  4.2 ± 0.2  4.2 ± 0.1 Ca²⁺ (mmol/L) 1.2 ± 0.1  1.2± 0.1  1.4 ± 0.1  1.4 ± 0.1 Glucose (mmol/L) 8.9 ± 0.6 10.2 ± 0.3 10.2 ±0.5 10.2 ± 0.7 % oxygen saturation 99.6 ± 0.1  99.8 ± 0.1 98.7 ± 1.299.7 ± 0.1 There were 4-6 support animals per group. All values areexpressed as mean ± SEM. *Drug was D-Asp D-Ala D-His D-Lys.

TABLE 4 Baseline systolic pressure and heart rate in the supportblood-perfused rat Subsequent Control values (t = 15 min aerobicperfusion) Treatment Group Systolic Pressure (mmHg) Heart Rate (bpm)Saline Control 96.4 ± 2.6 296.6 ± 9.8 Drug* 98.1 ± 3.8 297.6 ± 8.7 Allvalues are expressed as mean ± SEM. There were 6 support animals pergroup. *Drug was D-Asp D-Ala D-His D-Lys.

TABLE 5 Baseline left ventricular developed pressure (LVDP), heart rateand perfusion pressure in isolated blood-perfused rat hearts beforevarious interventions Control values (t = 20 min aerobic perfusion)Subsequent Heart Rate Perfusion Pressure Treatment Group LVDP (mmHg)(bpm) (mmHg) Saline Control 177.3 ± 10.6 236.6 ± 17.9 94.6 ± 7.5 Drug*177.2 ± 5.6  257.3 ± 29.7 99.6 ± 7.3 All values are expressed as mean ±SEM. There were 6 support animals per group. *Drug was D-Asp D-Ala D-HisD-Lys.

TABLE 6 Ischemic contracture during 30 min of global, zero flowischaemia Contracture Initiation Time-to-50% Peak Time-to-peak Group(min) (min) (mmHg) (min) Saline Control  9.2 ± 1.6 15.1 ± 0.5 93.7 ± 3.019.8 ± 0.6 Drug* 11.9 ± 1.7 16.5 ± 0.9 89.5 ± 3.0 21.7 ± 1.2 All valuesare expressed as the mean ± SEM. There were 6 hearts per group. *Drugwas D-Asp D-Ala D-His D-Lys.

Example 9 Testing of Asp Ala His Lys In A Brain Ischemia Model

Focal ischemic infarcts were made in mature male Wistar rats (270-300 g,Charles River Laboratories) as described previously (Koizumi et al, Jpn.J. Stroke, 8:1-8 (1986); Chen et al., J. Cereb.lood Flow Metab.,12(4):621-628 (1992)). Animals were allowed free access to food andwater before surgery. They were anesthetized with 3.5% halothane, andanesthesia was maintained with 1.0%-2.0% halothane in 70% N₂/30% O₂using a face mask. Rectal temperature was maintained at 37° C. duringsurgery using a feedback-regulated water heating system (YSI 73A rectalprobe, Fisher, connected to a K-20/64N aquatic blanket, HamiltonIndustries, Cincinnati, Ohio). Previous studies have shown that rectaland brain temperatures are identical during and after ischemia in thismodel (Chen et al., J. Cereb.lood Flow Metab., 12(4): 621-628 (1992)).The right femoral artery was cannulated with medical grade siliconetubing (Technical Products, Inc., Decatur, Ga.) for measurement of bloodgases and blood pressure, and the femoral vein was cannulated forinfusions. Cannulae were drawn through a subcutaneous tunnel and exitedthrough the dorsal neck. Arterial blood gases were measured in allanimals before and after ischemia.

For ischemia surgery, the right common carotid artery was exposed at itsbifurcation. A 4-0 nylon suture, with its tip rounded by heating over aflame, was then advanced 18.5-19.5 mm (depending on the animal's weight)from the external into the internal carotid artery and then through theintracranial carotid artery until the tip occluded the origin of themiddle cerebral artery (MCA). Animals were then allowed to awaken fromanesthesia. At 2 hours after MCA occlusion, they were re-anaesthetized,and intra-arterial sutures were withdrawn into the external carotidartery.

Beginning one minute prior to occlusion, animals received an intravenousinfusion of vehicle alone (control) or drug (D-Asp D-Ala D-His D-Lys) invehicle over one minute. The identity of the drug was unknown to theresearchers performing the experiments. It was supplied to theresearchers as a concentrated stock (16.67 mg/ml) in phosphate bufferedsaline, pH 7.4, and was stored it at −80° C. The drug was determined tobe biologically active prior to use by determining its ability to reducefree radical formation in vitro as described in Example 7. The stocksolution was thawed just prior to use, and a sufficient quantity wasadministered to give a dose of 20 mg/kg. At the end of the intravenousadministration of the drug or vehicle, the nylon suture was immediatelyadvanced to occlude the MCA. Following the 2 hours of occlusion of theMCA, the animals received a repeat intravenous infusion of drug orvehicle over one minute. At the end of the second infusion, the nylonsuture was immediately pulled back from occluding the MCA to allow forreperfusion. Also, after the second infusion, the animals werere-anesthetized, and the cannulae were removed. The animals werereturned to their home cages, where they were allowed free access tofood and water.

Animals were weighed before ischemia and before sacrifice. Aneurological examination, as described in Zea Longa et al., Stroke,20:84-91 (1989), was administered at 1 hour and at 24 hours afterreperfusion. Scoring was as follows: 0, normal; 1, failure to extendcontralateral (left) forepaw fully (amild focal neurologic deficit); 2,circling to the left (a moderate focal nuerologic deficit); 3, fallingto the left (a several focal neurologic deficit); and 4, no spontaneousgait and depressed level of consciousness.

Twenty-four hours after MCA occlusion, animals were anesthetized withketamine (44 mg/kg) and xylazine (13 mg/kg), both given intramuscularly,and perfused transcardially with heparinized saline, followed by 10%buffered formalin. The brains were removed and cut into 2-mm coronalslices using a rat brain matrix (Activational System, Inc., Warren,Mich.; a total of 7 slices). The slices were then embedded in paraffin,and 6-mm sections were cut from the anterior surface of each slice andstained with hematoxylin and eosin (H and E). Infarct volume wasdetermined using a computer-interfaced image analysis system (Global LabImage system, Data Translation, Marlboro, Mass.), using the “indirect”method (Swanson et al., J. Cerebral Blood Flow Metabol., 10:290-293(1990)): the area of intact regions of the ipsilateral (right)hemisphere and area of the intact contralateral (left) hemisphere weredetermined for each slice, the former was substracted from the latter tocalculate infract area per slice. Infarct areas were then summed andmultiplied by slice thickness to yield infarct volume per brain (inmm³).

The results are presented in Tables 7-10 below. Some of the data areexpressed as mean±S.E.M. Continuous data were analyzed by repeatedmeasures ANOVA and paired or unpaired two-tailed t-tests with Bonferronicorrection where appropriate. Non-continuous behavior date were analyzedby the Mann-Shitney U-test.

TABLE 7 Infarct Volume Treated* Control Animal Infarct Volume (mm³)Animal Infarct Volume (mm³) #1 11.5 #2 44.5 #3 14.7 #4 32.3 #5 43.3 #639.4 #7 10.9 #8 22.2 Mean 20.1 Mean 34.6 S.E.M. 7.7 S.E.M. 4.8 *Treatedwith D-Asp D-Ala D-His D-Lys

TABLE 8 Neurological Scale Treated* Control Animal Day 0^(a) Day 1^(b)Animal Day 0^(a) Day 1^(b) #1 2 1 #2 2 2 #3 2 1 #4 2 2 #5 2 2 #6 2 2 #72 1 #8 2 2 *Treated with D-Asp D-Ala D-His D-Lys ^(a)Day of ischemiasurgery ^(b)One day after ischemia surgery

TABLE 9 Body Weight Treated* Control Animal Day 0^(a) Day 1^(b) AnimalDay 0^(a) Day 1^(b) #1 300 272 #2 300 251 #3 300 266 #4 300 256 #5 295256 #6 278 230 #7 295 255 #8 300 250 *Treated with D-Asp D-Ala D-HisD-Lys ^(a)Day of ischemia surgery ^(b)One day after ischemia surgery

TABLE 10 Blood Gases And Blood Pressure 10 minutes before MCA occlusion10 minutes after MCA occlusion Ani- Ani- mal pH pCO₂ pO₂ BP^(c) mal pHpCO₂ pO₂ BP^(c) #1 7.437 38.5 117.1 96 #1 7.422 40.2 130.1 106 #3 7.43038.3 110.5 89 #3 7.423 40.1 130.1 98 #5 7.518 38.5 147.0 98 #5 7.47136.5 105.3 92 #7 7.423 33.4 97.4 86 #7 7.433 34.0 139.9 89 #2 7.401 35.5120.1 90 #2 7.423 40.1 130.1 103 #4 7.440 38.5 121.1 93 #4 7.421 39.5129.3 101 #6 7.425 35.9 110.0 88 #6 7.453 36.2 105.3 92 #8 7.417 39.5120.7 90 #8 7.428 36.9 111.0 93

Example 10 Inhibition of the Generation of ROS

The ability of the tetrapeptide L-Asp L-Ala L-His L-Lys [SEQ ID NO:1]and other peptides and compounds to inhibit the production of ROS wastested. The other peptides tested were: L-Asp L-Ala L-His L-Lys L-SerL-Glu L-Val L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID NO:3]; L-Ala L-HisL-Lys L-Ser L-Glu L-Val L-Ala L-His L-Arg L-Phe L-Lys [SEQ ID NO:4];L-His L-Lys L-Ser L-Glu L-Val L-Ala L-His L-Arg L-Phe L-Lys [SEQ IDNO:5]; and Acetylated-L-Asp L-Ala L-His L-Lys L-Ser L-Glu L-Val L-AlaL-His L-Arg L-Phe L-Lys [SEQ ID NO:6]. The peptides were obtained fromone or more companies that provide custom synthesis of peptides,including Ansynth Services, QCB, Genosys and Bowman Research. The othercompounds tested were histidine (Sigma Chemical Co.), catalase (SigmaChemical Co.), and superoxide dismutase (Sigma Chemical Co.).

1. Inhibition of Hydroxyl Radical Production

The hydroxyl radical is probably the most reactive oxygen-derivedspecies. The hydroxyl free radical is very energetic, short-lived andtoxic.

Some researchers suggest that the toxicity of hydrogen peroxide andsuperoxide radical may be due to their conversion to the hydroxyl freeradical. The superoxide radical can be directly converted to thehydroxyl radical via the Haber-Weiss reaction. Alternatively, it can beconverted to hydrogen peroxide which, in turn, is converted into thehydroxyl radical via the Fenton reaction. Both pathways require atransition metal, such as copper (Acworth and Bailey, The Handbook OfOxidative Metabolism (ESA, Inc. 1997)).

It is also known that copper, in the presence of ascorbate, produceshydroxyl radicals. The following reaction scheme has been suggested:

Ascorbate+2Cu²⁺→2Cu⁺+dehydroascorbate+2H⁺  (Eq. 1)

Cu⁺+O₂→O₂ ^(•−)+Cu²⁺  (Eq. 2)

Cu⁺+O₂ ^(•−)+2H⁺→Cu²⁺+H₂O₂  (Eq. 3)

Cu⁺+H₂O₂→OH⁻+OH^(•)+Cu²⁺  (Eq. 4)

Biaglow et al., Free Radic. Biol. Med., 22(7):1129-1138 (1997).

The ability of the compounds listed above to inhibit the generation ofhydroxyl radicals was tested as described in Gutteridge and Wilkins,Biochim. Biophys. Acta, 759:38-41 (1983). Briefly, Cu(II) and ascorbicacid were mixed causing the generation of hydroxyl radicals. Then,deoxyribose was added, and the hydroxyl radicals, if present, attackedthe deoxyribose to produce fragments. Heating the fragments at low pHproduced malonaldehyde that, upon the addition of 2-thiobarbituric acid(TBA), yielded a pink chromogen which was measuredspectrophotometrically at 532 nm. Thus, absorbance at 532 nm is ameasure of the damage to deoxyribose and, therefore, of hydroxyl radicalformation.

To perform the assay, CuCl₂ in buffer (20 mM KH₂PO₄ buffer, pH 7.4) andeither one of the test compounds in buffer or buffer alone were added totest tubes (final concentration of CuCl₂ was 10 μM). The test tubes wereincubated for 15 minutes at room temperature. Then, 0.5 mM ascorbic acidin buffer and 1.9 mM 2-deoxy-D-ribose in buffer were added to each testtube, and the test tubes were incubated for 1 hour at 37° C. Finally, 1ml of 1% (w/v) TBA in 50 mM NaOH and 1 ml of concentrated acetic acidwere added to each test tube, and the test tubes were incubated inboiling water for 15 minutes. After the test tubes had cooled for 15minutes, the absorbance at 532 nm was read.

It was found that the tetrapeptide L-Asp L-Ala L-His L-Lys [SEQ ID NO:1]caused complete inhibition of the formation of hydroxyl radicals in thisassay at tetrapeptide/copper ratios of 2:1 or higher.Tetrapeptide/copper ratios less than 2:1 were ineffective.

The results of a time course are presented in FIG. 15A. As can be seenin FIG. 15A, copper and ascorbate (no added peptide) producedTBA-reactive substances quickly and reached a maximum in 30 minutes. Thetetrapeptide at a tetrapeptide/copper ratio of 2:1 prevented allformation of TBA-reactive substances. Interestingly, the tetrapeptide ata tetrapeptide/copper ratio of 1:1 slowed the production of TBA-reactivesubstances. These data suggest that the tetrapeptide at a 1:1tetrapeptide/copper ratio is able to offer some protection from hydroxylradicals by binding copper which results in site-directed hydroxylattack on the tetrapeptide. Once enough of the tetrapeptide isdestroyed, then copper is released, which allows it to produce hydroxylradicals that attack the dexoyribose.

When the tetrapeptide at a tetrapeptide/copper ratio of 2:1 wasincubated for longer periods of time, its ability to prevent theformation of TBA-reactive substances slowly eroded. See FIG. 15B. As canbe seen from FIG. 15B, the production of TBA-reactive substances wasinhibited by 95% during the first 4 hours of incubation. By 24 hours,the level of inhibition had dropped to 50% and, by 48 hours, the levelof inhibition had dropped to 20%. These data suggest that TBA-reactivesubstances are still being produced even in the presence of thetetrapeptide. They also suggest that the tetrapeptide is being degradedduring the time course of the experiment. This degradation is more thanlikely due to the formation of free radicals in close proximity to thetetrapeptide/copper complex which attack and degrade the tetrapeptide,with release of the copper. Since free radicals, such as the hydroxylradical, are very reactive, they will attack the first electron richmolecule they come into contact with, which would be the tetrapeptide inthis case.

The effect of pH on the inhibition of hydroxyl radical formation by thetetrapeptide was tested at a tetrapeptide/copper ratio of 2:1. At thisratio, the tetrapeptide gave >95% inhibition of the formation ofTBA-reactive species at pH 7.0-8.5. These are physiological pH levelsand pH levels that would be expected during ischemia (acidosis occurs inischemic tissues). At pH 6.0, the tetrapeptide was ineffective atpreventing the formation of TBA-reactive species, possibly due to thereduced ability of the histidine to bind copper. The nitrogen atom onthe imidazole ring of histidine participates in binding copper with apKa of 6.0. Therefore, at a pH of 6.0, histidine is only able to bind50% of the copper. The other 50% of the copper would be unbound orloosely bound to the tetrapeptide by the other amino acids and would,therefore, be able to participate in the production of TBA-reactivespecies.

Histidine and several peptides with histidine in different positionswere tested at 1:1 and 2:1 peptide:copper ratios for their ability toinhibit the production of hydroxyl radicals. Also, a peptide having anacetylated aspartic acid (Ac-Asp) as the N-terminal amino acid was alsotested. The results are presented in Table 11. In Table 11, the %inhibition is the percent decrease in absorbance compared to bufferalone divided by the absorbance of the buffer alone.

As can be seen from the results in Table 11, the peptides with histidinein the second and third positions gave >95% inhibition at a 2:1peptide:copper ratio, while these peptides at a 1:1 peptide:copper ratiowere ineffective. Interestingly, at a 2:1 peptide:copper ratio, thepeptide with histidine in the first position and the peptide withacetylated aspartic acid as the N-terminal amino acid provided someprotection (about 47% and about 28% inhibition, respectively), althoughthis protection might be attributable to the histidine in the seventhand ninth positions, respectively, of these peptides. Histidine alone ata 2:1 histidine:copper ratio provided some protection (about 20%inhibition).

Catalase has been shown to prevent hydroxyl radical formation.Gutteridge and Wilkins, Biochim. Biophys. Acta, 759:38-41 (1983);Facchinetti et al., Cell. Molec. Neurobiol., 18(6):667-682 (1998);Samuni et al., Eur. J. Biochem., 137:119-124 (1983). Catalase (0-80 nM)was, therefore, tested in this assay, and it was found to prevent theformation of the pink chromogen (data not shown). This finding suggeststhat hydrogen peroxide is formed in this assay, since catalase breaksdown hydrogen peroxide to water and agrees with Equations 3 and 4 above.Catalase also prevents the formation of the pink chromogen when theL-Asp L-Ala L-His L-Lys [SEQ ID NO:1] tetrapeptide at atetrapeptide/copper ratio of 1:1 is present (data not shown). As shownabove, at this ratio, the copper is still able to participate in theredox reactions to produce hydroxyl radicals. These experiments showthat hydrogen peroxide is an important precursor to the formation of thehydroxyl radical.

TABLE 11 Absorbance Absorbance % Compound (Ratio)^(a) at 532 nm at 532nm Inhibition Copper only (buffer control) 0.767* 0.954 0Histindine/copper (2:1) 0.760 20.3 His Lys Ser Glu Val Ala His 0.71624.9 Arg Phe Lys^(b)/copper (1:1) His Lys Ser Glu Val Ala His 0.509 46.6Arg Phe Lys^(b)/copper (2:1) Ala His Lys Ser Glu Val Ala His 0.843 11.6Arg Phe Lys^(c)/copper (1:1) Ala His Lys Ser Glu Val Ala His 0.047 95.1Arg Phe Lys^(c)/copper (2:1) Asp Ala His Lys Ser Glu Val 0.645 13.2 AlaHis Arg Phe Lys^(d)/copper (1:1) Asp Ala His Lys Ser Glu Val 0.040 95.8Ala His Arg Phe Lys^(d)/copper (2:1) Ac-Asp Ala His Lys Ser Glu Val0.633 16.9 Ala His Arg Phe Lys^(e)/copper (1:1) Ac-Asp Ala His Lys SerGlu Val 0.692 27.5 Ala His Arg Phe Lys^(e)/copper (2:1) Asp Ala HisLys^(f)/copper (1:1) 0.751* 1.3 Asp Ala His Lys^(f)/copper (2:1) 0.029*96.2 ^(a)All amino acids are L-amino acids. ^(b)SEQ ID NO: 5 ^(c)SEQ IDNO: 4 ^(d)SEQ ID NO: 3 ^(e)SEQ ID NO: 6 ^(f)SEQ ID NO: 1 *Data takenfrom Table 2 in Example 7.

B. Assay For Superoxide Dismutase (SOD) Activity

The enzyme superoxide dismutase (SOD) is a naturally-occurring enzymewhich is responsible for the breakdown in the body of superoxide tohydrogen peroxide (similar to Equation 3). Hydrogen peroxide can then bedetoxified by catalase.

SOD was assayed for activity in the assay described in the previoussection and was found to have none (data not shown). This result is notsurprising since SOD actually converts superoxide radical into hydrogenperoxide. Hydrogen peroxide can then be converted into the hydroxylradical by reduced copper.

There are reports in the literature that copper complexes have SODactivity. Athar et al., Biochem. Mol. Biol. Int., 39(4):813-821 (1996);Ciuffi et al., Pharmacol Res., 38(4):279-287 (1998); Pogni et al., J.Inorg. Biochem., 73:157-165 (1999); Willingham and Sorenson, Biochem.Biophys. Res. Commun., 150(1):252-258 (1988); Konstantinova et al., FreeRad. Res. Comms., 12-13:215-220 (1991); Goldstein et al., J. Am. Chem.Soc., 112:6489-6492 (1990). This finding is not surprising since SODitself has copper in its active site.

The SOD activity of copper complexes of the tetrapeptide L-Asp L-AlaL-His L-Lys [SEQ ID NO: 1] was assayed. Superoxide radicals wereproduced using the xanthine oxidase assay of Beauchamp and Fridovich,Anal. Biochem., 44:276-287 (1971). Xanthine oxidase converts xanthineinto uric acid, with oxygen acting as an electron acceptor. This causessuperoxide radical to be produced. Superoxide radical is able to reducenitro blue tetrazolium (NBT). Reduced NBT has a λmax of 560 nm. It isknown that copper inhibits xanthine oxidase activity (Konstantinova etal., Free Rad. Res. Comms., 12-13:215-220 (1991)), so all experimentscontaining copper also contained ethylenediaminetetracetic acid (EDTA),a known copper chelator. The EDTA-copper complex was tested for SODactivity and was shown to have no SOD activity (data not shown).

To perform the assay for SOD activity, 0.1 mM xanthine (Sigma ChemicalCo.), 25 μM NBT (Sigma Chemical Co.), 50 mM sodium carbonate, and 1.2 μMEDTA (Sigma Chemical Co.), were mixed in a cuvette (all finalconcentrations, final pH 10.2). The reaction was started by the additionof various amounts of a tetrapeptide-copper complex (tetrapeptide/copperratios of 1:1 and 2:1) and 20 nM xanthine oxidase (Sigma Chemical Co.).The tetrapeptide-copper complex was prepared by mixing the tetrapeptideand copper (as CuCl₂) and allowing the mixture to incubate for 15minutes at room temperature immediately before addition to the cuvette.The samples were read at time 0 and every 60 seconds for five minutes at560 nm.

The complex of the tetrapeptide with copper at a ratio of 1:1 was shownto have SOD activity, as evidenced by inhibition of NBT reduction (seeFIG. 16). However, the complex was about 500 times less effective thanSOD itself, based on IC₅₀ values (amount that gives 50% inhibition) inthis assay. The complex of the tetrapeptide with copper at a ratio of2:1 was found to have no SOD activity (data not shown).

To verify that the 1:1 tetrapeptide-copper complex did not interferewith xanthine oxidase activity, uric acid production was measured at 295nm. Athar et al., Biochem. Mol. Biol. Int., 39(4):813-821 (1996); Ciuffiet al., Pharmacol Res., 38(4):279-287 (1998). This assay is similar tothe SOD assay, except that NBT is not present. Instead, uric acid isassayed at 295 nm every 60 seconds for 5 minutes. It was found that the1:1 tetrapeptide-copper complex only inhibited uric acid production by11% at a concentration of 600 nM (data not shown). Therefore, the 1:1tetrapeptide-copper complex has true SOD activity. Since superoxide isconverted to hydrogen peroxide by the complex, this could help toexplain why it is not effective at preventing hydroxyl radicalproduction.

Superoxide radical production was measured in solutions containing the1:1 or 2:1 tetrapeptide-copper complexes. The assay combined techniquesfrom the TBA assay and the xanthine oxidase assay. NBT was added to alltest tubes in order to quantitate its reduction by superoxide radical.The samples also contained ascorbate and copper and were incubated at37° C. At 5, 15, 30 and 60 minutes, the samples were removed from theincubator and read at 560 nm. The results are shown in FIG. 17. In thesample containing the 2:1 tetrapeptide-copper complex, NBT reductionincreased over time and reached a maximum at 30 minutes. The samplecontaining the 1:1 tetrapeptide-copper complex also showed an increasein NBT reduction, with a decreased maximum reached at 60 minutes. Thesedata suggest that superoxide accumulates in the sample containing the2:1 tetrapeptide-copper complex, while the 1:1 tetrapeptide-coppercomplex mimics superoxide dismutase.

The likely sequence of events that occurs in the production of hydroxylradicals is as follows:

O₂→O₂ ^(•−)→H₂O₂→OH^(•)  (Eq. 5).

It has already been shown that the 1:1 tetrapeptide-copper complex canconvert superoxide radical (O₂ ^(•−)) into hydrogen peroxide (H₂O₂).This is the SOD activity of the complex. The 2:1 tetrapeptide-coppercomplex cannot facilitate this conversion since the two molecules of thetetrapeptide fill all six coordination bonds of copper. This explainswhy the 2:1 tetrapeptide-copper complex is so effective because itinhibits the formation of hydrogen peroxide, which could in turn reactwith reduced copper to produce hydroxyl radicals via the Fentonreaction. The 1:1 tetrapeptide-copper complex also provides a valuableservice by eliminating the superoxide radical. Even though it produceshydrogen peroxide, most compartments of the human body have sufficientquantities of the enzyme catalase that can eliminate hydrogen peroxide.In the brain, however, catalase activity is reported to be minimal.Halliwell et al., Methods in Enzymol., 186:1-85 (1990). Therefore, thebrain is a particularly vulnerable organ during periods of ischemia,since copper is released due to the acidosis that accompanies ischemia.

C. Protection of DNA

DNA strand breaks were measured according to the method of Asaumi etal., Biochem. Mol. Biol. Int., 39(1):77-86 (1996). Briefly, 17 μg/ml ofplasmid pBR322 DNA was allowed to pre-incubate for 15 minutes at roomtemperature with 50 μM CuCl₂ and concentrations of the tetrapeptide of0-200 μM. Then, 2.5 mM ascorbate was added to each reaction, and themixture was incubated for 1 hour at 37° C. The total volume of themixture was 16 μL. Next, 3 μL of loading buffer containing 0.25% (w/v)bromophenol blue, 0.25% (w/v) xylene cyanole FF, and 40% (w/v) sucrosein water was added. The samples were separated by electrophoresis in a0.8% agarose gel for 90 minutes at 70 Volts. The gel was stained in1×TBE (Tris-Borate-EDTA buffer) containing 2 μg/ml ethidium bromide for30 minutes. The gel was then destained in 1×TBE for 5 minutes prior tophotographing the gel.

The results showed that the tetrapeptide was very effective atpreventing the formation of DNA strand breaks. See FIG. 18. Optimalprotective tetrapeptide:copper ratios were 2:1 and greater, sincesuperhelical circular DNA was still visible on the gel at these ratios.At a tetrapeptide:copper ratios of 1:1 or less, nicked circular DNA,linear DNA and more damaged DNA (smears) were visible.

Example 11 Reduction of the Damage Done to DNA by ROS

ROS damages DNA by causing strand breaks, base modifications, pointmutations, altered methylation patterns, and DNA-protein cross linking(Marnett, Carcinogenesis 21:361-370 (2000); Cerda et al., Mutat. Res.386:141-152 (1997)). Copper, iron, and other transition metals, in thepresence of reducing agents, catalyze the production of ROS such assuperoxide (O₂•), hydrogen peroxide (H₂O₂) and the hydroxyl radical(OH•) through both the Haber-Weiss and Fenton reactions (Stoewe et al.,Free Radic. Biol. Med. 3:97-105 (1987)). OH• is considered the mostreactive and damaging ROS and is capable of producing all the above DNAlesions (Marnett, Carcinogenesis 21:361-370 (2000)). Previousinvestigations have reported that OH• induced, single- and double-strandDNA breaks occur during site-specific copper ion reactions in vitro andduring excessive copper exposure in vivo (Chiu et al., Biochemistry34:2653-2661 (1995); Kim et al., Free Radic. Res. 33:81-89 (2000);Hayashi et al., Biochem. Biophys. Res. Comm. 276:174-178,doi:10.1006/bbrc.2000.3454 (2000)).

Telomeres, which are repeats of the hexanucleotide TTAGGG, exist at theends of DNA to form a “protective cap” against degradation, chromosomalrearrangement, and allow the replication of DNA without the loss ofgenetic information (Reddel, Carcinogenesis 21:477-484 (2000)). Theclassical theory of cellular aging, or senescence, involves the telomereend replication problem (Olovnikov, J. Theor. Biol. 41:181-190 (1973)).DNA polymerase is unable to replicate the terminal end of the laggingstrand during DNA replication resulting in the loss of 30-500 base pairs(Harley et al., Nature 345:458-460 (1990); von Zglinicki et al., Exp.Cell Res. 220:186-193, doi:10.1006/excr.1995.1305 (1995)). Somatic cellsare unable to replace these lost telomeric repeats, leading toprogressive telomere shortening during a cell's replicative life.Senescence is manifested when telomere length reaches a criticalthreshold (Reddel, Carcinogenesis 21:477-484 (2000)). Prematuresenescence has been documented in human fibroblasts exposed to oxidativestress (Chen et al., Proc. Natl. Acad. Sci. USA 91:4130-4134 (1994)).Examination of telomere length in fibroblasts after several populationdoublings under conditions of higher oxidative stress reveals shortenedtelomere lengths similar to senescence under normal conditions (vonZglinicki et al., Exp. Cell Res. 220:186-193, doi:10.1006/excr.1995.1305(1995)). These data suggest that ROS-induced DNA damage in the telomeresequence may play an important role in telomere shortening.

In this study, the ability of Asp Ala His Lys [SEQ ID NO:1] to protectDNA and telomeres from ROS damage induced by copper coupled withascorbic acid was examined.

A. Materials and Methods

Reagents: The synthetic D-analog of Asp Ala His Lys (D-Asp Ala His Lys)was obtained from Bowman Research Ltd. (Newport, Wales, UK). TeloTAGGTelomere Length Assay and X-ray film were purchased from Roche MolecularBiochemicals (Mannheim, Germany). DNeasy genomic isolation kits werepurchased from Qiagen (Valencia, Calif.). Hybond-N+nylon membrane wasordered from Amersham Pharmacia Biotech (Piscataway, N.J.). All otherchemicals were obtained from Sigma (St. Louis, Mo.).

DNA treatments: DNA strand breaks were measured using a modified methodof Asaumi (Asaumi et al., Biochem. Mol. Biol. Int. 39:77-86 (1996)).Raji cells, a Burkitt lymphoma derived cell line (obtained from AmericanType Culture Collection (ATCC), Rockville, Md., ascension numberCCL-86), were grown in Iscove's modified Dulbecco's medium (IMDM) with10% fetal calf serum (FCS) at 10% CO₂ and 37° C. Genomic DNA wasisolated using DNeasy spin columns (Qiagen) following the manufacturer'sprotocol. Then, 1 μg genomic DNA was incubated per reaction with CuCl₂,ascorbic acid, and/or the tetrapeptide in 10 mM sodium phosphate buffer,pH 7.4. Final concentrations were as follows: CuCl₂=10 μM, 25 μM, and 50μM; ascorbic acid=25 μM, 50 μM, and 100 μM; D-Asp Ala His Lys=50 μM, 100μM, and 200 μM. Total reaction volumes of 20 μl in 0.2 ml PCR tubes wereincubated at 37° C. for 2 hours. Following the incubation, strand breakswere visualized by immediately adding 5 μl of loading dye [0.25% (w/v)bromophenol blue and 40% (w/v) sucrose] and loading on a 0.5% trisacetic acid EDTA (TAE) agarose gel. Gels were then run at 70V for 90 mMand stained using 2 μg/ml ethidium bromide for 30 minutes. Prior tophotographing, gels were rinsed in TAE for 10 minutes.

Cell treatments: Raji cells were washed with PBS (10 mM phosphatebuffered saline; 138 mM NaCl; 2.7 mM KCl pH 7.4). Then, 1.5×10⁶ cellswere put into 5 ml PBS containing CuCl₂, ascorbic acid, and/or D-Asp AlaHis Lys. Final concentrations were as follows: CuCl₂=10 μM, 25 μM, and50 μM; ascorbic acid=100 μM, 250 μM, and 500 μM; D-Asp Ala His Lys=50μM, 100 μM, and 200 μM. The cells were then incubated at 37° C. for 2hours. Following the incubation, genomic DNA was isolated using DNeasycolumns. DNA damage was visualized by 0.5% TAE agarose gelelectrophoresis.

Telomere Length Assay: To examine telomere damage, the TeloTAGG TelomereLength Assay (Roche) was used according to manufacturer'srecommendations: digesting 1 μg of genomic DNA per reaction using HinfIand RSA I. Samples were then run on a 0.8% TAE agarose gel at 70V for 2hours. Southern blots were performed and probed using a digoxigenin(DIG) labeled telomere specific oligonucleotide. For cell treatedsamples, genomic DNA was used as described above. For DNA treatedsamples, reactions were setup as above, brought to 200 μl with PBS, andisolated using DNeasy columns prior to restriction digestions.

B. Results and Discussion

Copper ions, an essential part of chromatin (Dijkwel et al., J. CellSci. 84:53-67 (1986)), are present within DNA (Wacker et al., J. Biol.Chem. 234:3257-3262 (1959)) and may participate in oxidative DNA damage(Chiu et al., Biochemistry 34:2653-2661 (1995); Hayashi et al., Biochem.Biophys. Res. Comm. 276:174-178, doi:10.1006/bbrc.2000.3454 (2000);Kagawa et al., J. Biol. Chem. 266:20175-20184 (1991)). In the presenceof ascorbate or other reducing agents, copper can lead to the productionof ROS by catalyzing the following reactions (Biaglow et al., FreeRadic. Biol. Med. 22:1129-1138 (1997)):

1) 2 Cu₂ ²⁺+ascorbate→2 Cu⁺+dehydroascorbate+2H⁺

2) Cu⁺+O₂→O₂•⁻+Cu²⁺

3) Cu⁺+O₂•⁻+2H⁺→Cu²⁺+H₂O₂

4) Cu⁺+H₂O₂→OH⁻+OH•+Cu²⁺

While iron is found at higher concentrations physiologically, oxidationby copper and H₂O₂ is 50 times faster than iron (Stoewe et al., FreeRadic. Biol. Med. 3:97-105 (1987); Halliwell J. Neurochem. 59:1609-1623(1992)). Due to the negative charge of the sugar phosphate backbone,cations can loosely bind DNA. Site-specific binding of copper ionswithin base pairs may be important to the regulation of DNA biosynthesis(Minchenkova et al., Biopolymers 5:615-625 (1967)). Unlikeiron-catalyzed reactions, OH• scavengers do not prevent copper-mediatedoxidative damage suggesting that oxidative DNA damage occurs in closeproximity to the copper ions (Oikawa et al., Biochim. Biophys. Acta1399:19-30 (1998)). The reactivity of OH• is so great that, presumably,OH• interactions only occur at or near the site of OH• production(Marnett, Carcinogenesis, 21, 361-370 (2000)). Oikawa, et. al., (Oikawaet al., Biochim. Biophys. Acta 1399:19-30 (1998)) have shown that thefollowing copper-mediated ROS reaction also occurs, and that theresulting DNA-copper-peroxide complex may be even more damaging to DNAthan OH•:

Cu⁺+H₂O₂→Cu⁺OOH+H⁺

As expected, the results of the above-described experiments showed thatcopper and ascorbic acid alone were unable to cause strand breaks. WhenCuCl₂ and ascorbic acid were combined, a dose dependent accumulation oflower molecular weight DNA fragments was seen, the result of doublestrand breaks. These double strand breaks were attenuated by D-Asp AlaHis Lys in a dose dependent manner (FIG. 20). At molar ratios of 1:1 (50μM copper to 50 μM D-Asp Ala His Lys) and 1:2, some strand breaks wereapparent. By elevating the ratio to 1:4, no strand breaks were detected.Similar results were observed in Raji cells treated with copper andascorbic acid (FIG. 21). A lower ratio of 1:2 (copper to D-Asp Ala HisLys) provided complete protection to DNA in cell samples. It isreasonable to expect that DNA samples would require higher D-Asp Ala HisLys levels due to competition for copper with DNA and proximal OH•attack. The separation of DNA and copper would be critical in thesesamples necessitating the need for elevated D-Asp Ala His Lys. In cellsamples, damage would be attributable to H₂O₂.H₂O₂ is freely diffusible,can penetrate to the nucleus, and has been shown to damage DNA infibroblasts (Chen et al., Proc. Natl. Acad. Sci. USA 91:4130-4134(1994); von Zglinicki et al., Free Radic. Biol. Med. 28:64-74 (2000)).Entrance of H₂O₂ into the cell may lead either to the formation of DNAperoxide complexes with native metals or to the release of sequesteredmetal stores that, combined with endogenous reducing agents (reducedglutathione (GSH), reduced nicotinamide dinucleotide (NADH), andascorbic acid), would drive the production of OH•. One possiblemechanism of D-Asp Ala His Lys protection would be the chelation ofcopper ions, thereby preventing production of OH• and H₂O₂. Another modeof protection may be the formation of D-Asp Ala His Lys-copper-peroxidecomplexes which would absorb the OH• damage rather than DNA, “mop-up”peroxides, and perhaps, in cell samples, keep H₂O₂ outside the cell.

Prior reports suggest that oxidative DNA damage may be directed at G-Crich areas, including telomeres. Rodriguez, et. al., reported thatcopper induced ROS damage primarily targeted DNA guanine (Rodriguez etal., Cancer Res. 57:2394-2403 (1997)). Strong, preferential binding ofCu (II) to the G-C pair has been reported at the N-7 and O-6 of theguanine bases plus the N-3 of cytosine (Kagawa et al., J. Biol. Chem.266:20175-20184 (1991)). DNA peroxides complexes formed at thesepositions are believed to direct OH• attack to adjacent bases (Oikawa etal., Biochim. Biophys. Acta 1399:19-30 (1998)). In addition, GGG intelomeric DNA has been shown to be sensitive to copper mediated ROSdamage (Oikawa et al., FEBS Lett. 453:365-368 (1999)).

Examination of the telomere in the genomic DNA samples in the presentstudy showed double strand breaks in response to oxidative stress. DNAsamples examined by Southern blot showed severely depleted and shortenedtelomere sequences (FIG. 22). Cell treatments showed damage to thetelomere with some conservation of the sequence, even at the highestlevels of copper and ascorbic acid used (FIG. 23), which may beattributed to ROS production outside the cells with the DNA shelteredinside the nucleus. D-Asp Ala His Lys protected the telomere fromcopper-mediated damage in these samples.

In addition to the double strand breaks detected in the experiments,other DNA lesions may be involved in ROS disease processes. Somecations, including copper, bound loosely to the phosphate backbone havebeen implicated in strand breaks while those coordinated in the helixcause base modifications (Marnett, Carcinogenesis 21:361-370 (2000);Rodriguez et al., Cancer Res. 57:2394-2403 (1997)). Episodes ofincreased copper and oxidative stress may direct DNA damage to G-C richareas. In addition to telomeres, G-C rich areas exist at the 5′ end ofmany genes (Bird, Nature 321:209-213 (1986)) hinting toward a site ofoxidative damage involved in gene regulation. 8-Oxo-deoxyguanosine(8-oxo-dG) is a common DNA adduct produced by ROS, which can result inG→T point mutations widely seen in mutated oncogenes (Marnett,Carcinogenesis 21:361-370 (2000)). Conditions such as acidosis occurringduring myocardial ischemia or alterations of ceruloplasmin have beenshown to mobilize free copper to catalyze local oxidative tissue and DNAdamage (Kim et al., Free Radic. Res. 33:81-89 (2000); Chevion et al.,Proc. Natl. Acad. Sci. USA 90:1102-1106 (1993)). Levels of 8-oxo-dG arereported to be three to four times higher in the DNA of ischemic rathearts than in controls (You et al., J. Mol. Cell. Cardiol.32:1053-1059, doi:10.1006/jmcc.2000.1142 (2000)). In addition, chronicinflammation can produce areas of localized oxidative damage.Inflammatory cells, such as macrophages and neutrophils, release ROSthat have been shown to damage the DNA of nearby cells (Shacter et al.,Carcinogenesis 9:2297-2304 (1988)). Nitric oxide and superoxide releasedfrom activated leukocytes can lead to the production of peroxynitrite,which is more reactive with 8-oxo-dG than unmodified bases and possiblyexacerbates the damage (Marnett, Carcinogenesis 21:361-370 (2000)).

C. Summary

Both DNA and the telomeric sequence are susceptible to copper-mediatedROS damage, particularly damage attributed to hydroxyl radicals. In thisstudy, ROS-induced DNA double strand breaks and telomere shortening wereproduced by exposure to copper and ascorbic acid. D-Asp-Ala-His-Lys, acopper chelating tetrapeptide D-analog of the N-terminus of humanalbumin, attenuated DNA strand breaks in a dose dependent manner. TheD-tetrapeptide, at a ratio of 4:1 (peptide:Cu), provided completeprotection of isolated DNA and, at a ratio of 2:1 (peptide:Cu),completely protected Raji Burkitt cells' DNA exposed tocopper/ascorbate. Southern blots of DNA treated with copper/ascorbateshowed severe depletion and shortening of telomeres with someconservation of telomere sequences. The D-tetrapeptide provided completetelomere length protection at a ratio of 2:1 (peptide:Cu). While theexact mechanisms for ROS DNA damage have yet to be fully elucidated,D-Asp Ala His Lys inhibited copper-induced DNA double-strand breaks byROS in both genomic DNA and in the telomere sequence.

Example 12 Inhibition of Angiogenesis

SPF research-grade fertilized eggs were obtained from Charles RiverLaboratories (800-772-3721). The eggs were candled to determine theposition of the yolk and to mark the air cell with a pencil. Understerile conditions, a small hole was drilled in the shell using a microhand drill. The eggs were divided randomly into four groups, six eggsper group:

A—no injection;

B—100 μl of 7.0 mg/ml the peptide Asp Ala His Lys [SEQ ID NO:1]injected;

C—100 μl of 3.5 mg/ml the peptide Asp Ala His Lys [SEQ ID NO:1]injected; and

D—100 μl of water injected.

Injections were made into the yolk using a 0.5 ml syringe. The eggs werelabeled at the time of injection with their group designations using apencil. The holes in the injected eggs were sealed with candle wax, andthe eggs were incubated in a Hova-Bator incubator (obtained from G.Q.F.Mfg. Co., Savannah, Ga.) set to 38° C. and 60-70% humidity (monitored byhygrometer inside incubator) for seven days. The eggs were incubated intrays with the small side down at a 30° angle. The eggs were turned sixtimes per day to allow proper development. The eggs were candled everyday for no more than 30 minutes total time out of the incubator, and anyvascular development was noted.

At the end of the seven-day incubation period, a window was opened inthe shell above the air cell using the following technique. The shellwas cracked gently above the air cell using forceps. A few flakes ofshell were removed with the forceps. The shell above the contents of theegg was removed using a pair of scissors. A drop of saline was placed onthe opaque inner membrane. A morphometric analysis of angiogenesis inthe chorioallantoic membranes was carried out, and the results aresummarized in Table 12 below and were documented by digital photography.

TABLE 12 No. Eggs Exhibiting Group Total No. Eggs Angiogenesis A 6 4 B 62 C 6 1 D 6 5Nine of the twelve eggs injected with the peptide (groups B and C) didnot develop a vascular system, whereas only three of twelve control eggs(groups A and D) failed to develop a vascular system. Statisticalanalysis of control eggs (groups A and D) versus treated eggs (groups Band C) showed that the difference in vascular development wasstatistically significant.

Example 13 Inhibition of IL-8 Release

Interleukin 8 (IL-8) is a pro-inflammatory cytokine and a potentchemoattractant and activator of neutrophils. It has also been reportedto be a chemoattractant and activator of T-lymphocytes and eosinophils.IL-8 is produced by immune cells (including lymphocytes, neutrophils,monocytes and macrophages), fibroblasts and epithelial cells. Reportsindicate an important role for IL-8 in the pathogenesis of respiratoryviral infections, asthma, bronchitis, emphysema, cystic fibrosis, acuterespiratory distress syndrome, sepsis, multiple organ dysfunctionsyndrome, and other inflammatory disorders.

The IL-8 release by Jurkat cells (American Type Culture Collection(ATCC), Rockville, Md.) exposed to copper and ascorbic acid (to produceROS—see Examples 7, 10 and 11) was investigated. To do so, 1×10⁶ Jurkatcells were incubated at 37° C. and 5% CO₂ in 0.5 ml IMDM medium (ATCC)(serum-free) with insulin transferin selenite solution (ITSS; Sigma) for24 hours with the following additives.

Experiment 1:

-   -   a. None (control);    -   b. Asp Ala His Lys [SEQ ID NO:1] (“DAHK”)—200 μM and ascorbic        acid—500 μM;    -   c. CuCl₂—10 μM and ascorbic acid—500 μM;    -   d. CuCl₂—25 μM and ascorbic acid—500 μM;    -   e. CuCl₂—50 μM and ascorbic acid—500 μM;    -   f. CuCl₂—100 μM and ascorbic acid—500 μM;    -   g. CuCl₂—50 μM and DAHK—50 μM and ascorbic acid—500 μM;    -   h. CuCl₂—50 μM and DAHK—100 μM and ascorbic acid—500 μM; and    -   i. CuCl₂—50 μM and DAHK—200 μM and ascorbic acid—500 μM.

Experiment 2:

-   -   a. None (control);    -   b. CuCl₂—100 μM;    -   c. DAHK—200 μM and ascorbic acid—500 μM;    -   d. CuCl₂—25 μM and ascorbic acid—500 μM;    -   e. CuCl₂—50 μM and ascorbic acid—500 μM;    -   f. CuCl₂—100 μM and ascorbic acid—500 μM;    -   g. CuCl₂—50 μM and DAHK—50 μM and ascorbic acid—500 μM;    -   h. CuCl₂—50 μM and DAHK—100 μM and ascorbic acid—500 μM; and    -   i. CuCl₂—50 μM and DAHK—200 μM and ascorbic acid—500 μM.

Experiment 3:

-   -   a. None (control);    -   b. CuCl₂—100 μM;    -   c. DAHK—400 μM and ascorbic acid—250 μM;    -   d. CuCl₂—25 μM and ascorbic acid—250 μM;    -   e. CuCl₂—50 μM and ascorbic acid—250 μM;    -   f. CuCl₂—100 μM and ascorbic acid—250 μM;    -   h. CuCl₂—100 μM and DAHK—200 μM and ascorbic acid—250 μM; and    -   i. CuCl₂—100 μM and DAHK—400 μM and ascorbic acid—250 μM.

After the 24-hour incubation, supernatants were collected and theconcentration of IL-8 in each supernatant was determined by an ELISAusing human IL-8 matched pair antibodies (Endogen, Cambridge, Mass.).The ELISA was performed using an ELISA kit from Endogen, Cambridge,Mass. according to the manufacturer's instructions with the followingexceptions: (1) coating antibody at 1 μg/ml; (2) detecting antibody 30ng/ml; StrepAvidin HRP diluted 1:32,000.

The results are presented in FIG. 24A (Experiment 1), FIG. 24B(Experiment 2), and FIG. 24 C (Experiment 3). As can be seen, copper andascorbic acid caused the release of Il-8 from the cells in adose-dependent manner. As can also be seen, DAHK inhibited the releaseof IL-8, with the best results being obtained with an 8:1 DAHK:Cu ratio.

Example 14 Inhibition of Oxidation of CoA

Coenzyme A (CoA) is essential for acetylation reactions in the body and,as a consequence, plays a critical role in the metabolism ofcarbohydrates and fatty acids. CoA can be oxidized to a disulfide whichcannot participate in acetylation reactions. As a result, metabolism andenergy utilization are inhibited.

In this example, it was investigated whether Cu(II) could oxidize CoAand, if so, whether the tetrapeptide Asp Ala His Lys [SEQ ID NO:1](Bowman Research, Inc., United Kingdom) could protect CoA (Sigma) fromoxidation by Cu(II). The experimental setup and results are presented inTable 13 below. All of the ingredients were added simultaneously and,after a 15-minute incubation, absorbance at 412 nm (A412) was measured.Free thiol groups were measured using DTNB. DTNB is dithionitrobenzoicacid (Sigma).

TABLE 13 1 2 3 4 5 6 7 Asp Ala 50 μl 50 μl 50 μl 50 μl 50 μl His Lys(190 μM) (190 μM) (190 μM) (190 μM) (190 μM) (2 mM) CoA 50 μl 50 μl 50ul 50 μl 50 μl (2 mM) (190 μM) (190 μM) (190 μM) (190 μM) (190 μM) CuCl₂100 μl 100 μl 100 μl 50 μl (1 mM) (190 μM) (190 μM) (190 μM) (85 μM)Tris 200 μl 300 μl 250 μl 350 μl 350 μl 250 μl 250 μl buffer, 50 mM, pH8.0 DTNB 125 μl 125 μl 125 μl 125 μl 125 μl 125 μl 125 μl (3 mM) A4120.279 1.119 0.127 0.888 0.142 0.113 1.111

As can be seen from Table 13, Cu(II) oxidized CoA. As can also be seen,the tetrapeptide at a 1:1 tetrapeptide:Cu(II) ratio provided someprotection of CoA, and the tetrapeptide at a 2:1 tetrapeptide:Cu(II)ratio provided 100% protection.

1.-418. (canceled)
 419. A method of reducing the damage done by reactiveoxygen species (ROS) in an animal in need thereof comprisingadministering to the animal an effective amount of a metal-bindingpeptide which does not have a metal ion bound to it or of aphysiologically-acceptable salt of the peptide, the sequence of thepeptide being:P₁-P₂, wherein: P₁ is: Xaa₁ Xaa₂ His or Xaa₁ Xaa₂ His Xaa₃, the P₁portion of the peptide being linear; Xaa₁ is the N-terminal amino acidof the peptide, the only substituents on the α-amino group of Xaa₁ arehydrogen, and Xaa₁ is glycine, alanine, valine, leucine, isoleucine,serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine,lysine, hydroxylysine, histidine, arginine, ornithine, phenylalanine,tyrosine, tryptophan, cysteine, methionine, or α-hydroxymethylserine;Xaa₂ is alanine, β-alanine, valine, leucine, isoleucine, serine,threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine,hydroxylysine, histidine, arginine, ornithine, phenylalanine, tyrosine,tryptophan, cysteine, methionine, or α-hydroxymethylserine; Xaa₃ isglycine, alanine, valine, lysine, arginine, ornithine, aspartic acid,glutamic acid, asparagine, glutamine or tryptophan; and P₂ is an aminoacid sequence which comprises the sequence of a Cu(I) binding site, andP₂ contains no more than 10 amino acids.
 420. The method of claim 419wherein: Xaa₁ is glycine, alanine, valine, leucine, isoleucine, serine,threonine, aspartic acid, glutamic acid, lysine, hydroxylysine,histidine, arginine, or α-hydroxymethylserine, and Xaa₂ is alanine,valine, leucine, isoleucine, threonine, serine, asparagine, glutamine,cysteine, methionine, lysine, hydroxylysine, histidine, arginine, orα-hydroxymethylserine.
 421. The method of claim 419 wherein Xaa₁ isaspartic acid, glutamic acid, arginine, threonine orα-hydroxymethylserine.
 422. The method of claim 419 wherein Xaa₂ isalanine, valine, leucine, isoleucine, threonine, serine, asparagine,methionine, histidine or α-hydroxymethylserine.
 423. The method of claim419 wherein Xaa₃ is lysine.
 424. The method of claim 419 wherein: Xaa₁is aspartic acid, glutamic acid, arginine, lysine, threonine, serine orα-hydroxymethylserine, Xaa₂ is alanine, valine, leucine, isoleucine,threonine, serine, asparagine, methionine, histidine orα-hydroxymethylserine, and Xaa₃, when present, is lysine.
 425. Themethod of claim 424 wherein Xaa₁ is aspartic acid or glutamic acid andXaa₂ is alanine, valine, leucine, isoleucine, threonine, serine orα-hydroxymethylserine.
 426. The method of claim 425 wherein Xaa₂ isalanine, valine, leucine or isoleucine.
 427. The method of claim 426wherein P₁ is Asp Ala His or Asp Ala His Lys.
 428. The method of claim427 wherein P₁ is Asp Ala His Lys.
 429. The method of claim 424 whereinXaa₁ is arginine, lysine, threonine, serine or α-hydroxymethylserine,and Xaa₂ is alanine, valine, leucine, isoleucine, threonine, serine orα-hydroxymethylserine.
 430. The method of claim 429 wherein P₁ is ThrLeu His, HMS HMS His or Arg Thr His.
 431. The method of claim 419wherein P₂ comprises one of the following sequences: Met Xaa₄ Met, MetXaa₄ Xaa₄ Met, Cys Cys, Cys Xaa₄ Cys, Cys Xaa₄ Xaa₄ Cys, Met Xaa₄ CysXaa₄ Xaa₄ Cys, Gly Met Xaa₄ Cys Xaa₄ Xaa₄ Cys [SEQ ID NO:7], Gly Met ThrCys Xaa₄ Xaa₄ Cys [SEQ ID NO:8], Gly Met Thr Cys Ala Asn Cys [SEQ IDNO:9], or δ-Glu Cys Gly; wherein Xaa₄ is any amino acid.
 432. The methodof claim 431 wherein P₂ is Gly Met Thr Cys Ala Asn Cys [SEQ ID NO:9].433. The method of claim 419 wherein at least one of the amino acids ofP₁ other than β-alanine or glycine, when present, is a D-amino acid.434. The method of claim 419 or 433 wherein at least one of the aminoacids of P₂ other than β-alanine or glycine, when present, is a D-aminoacid.
 435. The method of claim 419 wherein the terminal —COOH of P₁-P₂is substituted to produce —COR₂, wherein R₂ is —NH₂, —NHR₁, —N(R₁)₂,—OR₁, or —R₁, wherein R₁ is an alkyl, aryl or heteroaryl.
 436. Themethod of claim 431 wherein P₁ is Asp Ala His or Asp Ala His Lys. 437.The method of claim 432 wherein P₁ is Asp Ala His or Asp Ala His Lys.438. The method of claim 419 wherein the peptide is administered to theanimal because of the need to reperfuse an ischemic tissue or organ ofthe animal.
 439. The method of claim 438 wherein the animal is sufferingfrom cerebrovascular ischemia and the ischemic tissue is located in thebrain of the animal.
 440. The method of claim 438 wherein the animal issuffering from cardiovascular ischemia and the ischemic tissue islocated in the heart of the animal.
 441. The method of claim 438 whereinthe peptide is administered prior to reperfusion, simultaneously withreperfusion, after reperfusion, or combinations thereof.
 442. The methodof claim 419 wherein the peptide is administered to the animal becauseit is suffering from a neurological trauma.
 443. The method of claim 419wherein the peptide is administered to the animal because it issuffering from a neurodegenerative disease.
 444. The method of claim 419wherein the peptide is administered to the animal to reduce the damagedone by ROS to its DNA.
 445. The method of claim 419 wherein the peptideis administered to the animal because it is suffering from inflammation.446. The method of claim 419 wherein the peptide is administered to theanimal because it is suffering from a disease or condition involvingacidosis.
 447. The method of claim 419 wherein the peptide isadministered to the animal because it is suffering from sepsis.
 448. Themethod of claim 419 wherein the peptide is administeredprophylactically.
 449. The method of claim 448 wherein the peptide isadministered to an animal exhibiting symptoms of possiblecerebrovascular ischemia or possible cardiovascular ischemia while theanimal is being diagnosed.
 450. The method of claim 448 wherein thepeptide is administered to an animal prior to surgery, during surgery,after surgery, or combinations thereof.
 451. The method of claim 450wherein the surgery is open-heart surgery or surgery to transplant anorgan into the animal.
 452. The method of claim 448 wherein the peptideis administered to an animal prior to radiation therapy, duringradiation therapy, after radiation therapy, or combinations thereof.